The stoichiometry of P2X2/6 receptor heteromers depends on relative subunit expression levels

Fast synaptic transmission involves the operation of ionotropic receptors, which are often composed of at least two types of subunit. We have developed a method, based on atomic force microscopy imaging to determine the stoichiometry and subunit arrangement within ionotropic receptors. We showed recently that the P2X(2) receptor for ATP is expressed as a trimer but that the P2X(6) subunit is unable to oligomerize. In this study we addressed the subunit stoichiometry of heteromers containing both P2X(2) and P2X(6) subunits. We transfected tsA 201 cells with both P2X(2) and P2X(6) subunits, bearing different epitope tags. We manipulated the transfection conditions so that either P2X(2) or P2X(6) was the predominant subunit expressed. By atomic force microscopy imaging of isolated receptors decorated with antiepitope antibodies, we demonstrate that when expression of the P2X(2) subunit predominates, the receptors contain primarily 2 x P2X(2) subunits and 1 x P2X(6) subunit. In contrast, when the P2X(6) subunit predominates, the subunit stoichiometry of the receptors is reversed. Our results show that the composition of P2X receptor heteromers is plastic and dependent on the relative subunit expression levels. We suggest that this property of receptor assembly might introduce an additional layer of subtlety into P2X receptor signaling

Separating and visualising protein assemblies by means of preparative mass spectrometry and microscopy

a b s t r a c t Many multi-protein assemblies exhibit characteristics which hamper their structural and dynamical characterization. These impediments include low copy number, heterogeneity, polydispersity, hydrophobicity, and intrinsic disorder. It is becoming increasingly apparent that both novel and hybrid structural biology approaches need to be developed to tackle the most challenging targets. Nanoelectrospray mass spectrometry has matured over the last decade to enable the elucidation of connectivity and composition of large protein assemblies. Moreover, comparing mass spectrometry data with transmission electron microscopy images has enabled the mapping of subunits within topological models. Here we describe a preparative form of mass spectrometry designed to isolate specific protein complexes from within a heterogeneous ensemble, and to 'soft-land' these target complexes for ex situ imaging. By building a retractable probe incorporating a versatile target holder, and modifying the ion optics of a commercial mass spectrometer, we show that we can steer the macromolecular ion beam onto a target for imaging by means of transmission electron microscopy and atomic force microscopy. Our data for the tetradecameric chaperonin GroEL show that not only are the molecular volumes of the landed particles consistent with the overall dimensions of the complex, but also that their gross topological features can be maintained

History of oceanic front development in the New Zealand sector of the Southern Ocean during the Cenozoic--a synthesis

The New Zealand sector of the Southern Ocean (NZSSO) has opened about the Indian-Pacific spreading ridge throughout the Cenozoic. Today the NZSSO is characterised by broad zonal belts of antarctic (cold), subantarctic (cool), and subtropical (warm) surface-water masses separated by prominent oceanic fronts: the Subtropical Front (STF) c. 43deg.S, Subantarctic Front (SAF) c. 50deg.S, and Antarctic Polar Front (AAPF) c. 60deg.S. Despite a meagre database, the broad pattern of Cenozoic evolution of these fronts is reviewed from the results of Deep Sea Drilling Project-based studies of sediment facies, microfossil assemblages and diversity, and stable isotope records, as well as from evidence in onland New Zealand Cenozoic sequences. Results are depicted schematically on seven paleogeographic maps covering the NZSSO at 10 m.y. intervals through the Cenozoic. During the Paleocene and most of the Eocene (65-35 Ma), the entire NZSSO was under the influence of warm to cool subtropical waters, with no detectable oceanic fronts. In the latest Eocene (c. 35 Ma), a proto-STF is shown separating subantarctic and subtropical waters offshore from Antarctica, near 65deg.S paleolatitude. During the earliest Oligocene, this front was displaced northwards by development of an AAPF following major global cooling and biotic turnover associated with ice sheet expansion to sea level on East Antarctica. Early Oligocene full opening (c. 31 Ma) of the Tasmanian gateway initiated vigorous proto-circum-Antarctic flow of cold/cool waters, possibly through a West Antarctic seaway linking the southern Pacific and Atlantic Oceans, including detached northwards "jetting" onto the New Zealand plateau where condensation and unconformity development was widespread in cool-water carbonate facies. Since this time, a broad tripartite division of antarctic, subantarctic, and subtropical waters has existed in the NZSSO, including possible development of a proto-SAF within the subantarctic belt. In the Early-early Middle Miocene (25-15 Ma), warm subtropical waters expanded southwards into the northern NZSSO, possibly associated with reduced ice volume on East Antarctica but particularly with restriction of the Indonesian gateway and redirection of intensified warm surface flows southwards into the Tasman Sea, as well as complete opening of the Drake gateway by 23 Ma allowing more complete decoupling of cool circum-Antarctic flow from the subtropical waters. During the late Middle-Late Miocene (15-5 Ma), both the STF and SAF proper were established in their present relative positions across and about the Campbell Plateau, respectively, accompanying renewed ice buildup on East Antarctica and formation of a permanent ice sheet on West Antarctica, as well as generally more expansive and intensified circum-Antarctic flow. The ultimate control on the history of oceanic front development in the NZSSO has been plate tectonics through its influence on the paleogeographic changes of the Australian-New Zealand-Antarctic continents and their intervening oceanic basins, the timing of opening and closing of critical seaways, the potential for submarine ridges and plateaus to exert some bathymetric control on the location of fronts, and the evolving ice budget on the Antarctic continent. The broad trends of the Cenozoic climate curve for New Zealand deduced from fossil evidence in the uplifted marine sedimentary record correspond well to the principal paleoceanographic events controlling the evolution and migration of the oceanic fronts in the NZSSO

Nanoscale Mobility of the Apo State and TARP Stoichiometry Dictate the Gating Behavior of Alternatively Spliced AMPA Receptors.

Neurotransmitter-gated ion channels are allosteric proteins that switch on and off in response to agonist binding. Most studies have focused on the agonist-bound, activated channel while assigning a lesser role to the apo or resting state. Here, we show that nanoscale mobility of resting α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptors (AMPA receptors) predetermines responsiveness to neurotransmitter, allosteric anions and TARP auxiliary subunits. Mobility at rest is regulated by alternative splicing of the flip/flop cassette of the ligand-binding domain, which controls motions in the distant AMPA receptor N-terminal domain (NTD). Flip variants promote moderate NTD movement, which establishes slower channel desensitization and robust regulation by anions and auxiliary subunits. In contrast, greater NTD mobility imparted by the flop cassette acts as a master switch to override allosteric regulation. In AMPA receptor heteromers, TARP stoichiometry further modifies these actions of the flip/flop cassette generating two functionally distinct classes of partially and fully TARPed receptors typical of cerebellar stellate and Purkinje cells

A multidrug ABC transporter with a taste for salt.

BACKGROUND: LmrA is a multidrug ATP-binding cassette (ABC) transporter from Lactococcus lactis with no known physiological substrate, which can transport a wide range of chemotherapeutic agents and toxins from the cell. The protein can functionally replace the human homologue ABCB1 (also termed multidrug resistance P-glycoprotein MDR1) in lung fibroblast cells. Even though LmrA mediates ATP-dependent transport, it can use the proton-motive force to transport substrates, such as ethidium bromide, across the membrane by a reversible, H(+)-dependent, secondary-active transport reaction. The mechanism and physiological context of this reaction are not known. METHODOLOGY/PRINCIPAL FINDINGS: We examined ion transport by LmrA in electrophysiological experiments and in transport studies using radioactive ions and fluorescent ion-selective probes. Here we show that LmrA itself can transport NaCl by a similar secondary-active mechanism as observed for ethidium bromide, by mediating apparent H(+)-Na(+)-Cl(-) symport. Remarkably, LmrA activity significantly enhances survival of high-salt adapted lactococcal cells during ionic downshift. CONCLUSIONS/SIGNIFICANCE: The observations on H(+)-Na(+)-Cl(-) co-transport substantiate earlier suggestions of H(+)-coupled transport by LmrA, and indicate a novel link between the activity of LmrA and salt stress. Our findings demonstrate the relevance of investigations into the bioenergetics of substrate translocation by ABC transporters for our understanding of fundamental mechanisms in this superfamily. This study represents the first use of electrophysiological techniques to analyze substrate transport by a purified multidrug transporter

Standalone vertex finding in the ATLAS muon spectrometer

A dedicated reconstruction algorithm to find decay vertices in the ATLAS muon spectrometer is presented. The algorithm searches the region just upstream of or inside the muon spectrometer volume for multi-particle vertices that originate from the decay of particles with long decay paths. The performance of the algorithm is evaluated using both a sample of simulated Higgs boson events, in which the Higgs boson decays to long-lived neutral particles that in turn decay to bbar b final states, and pp collision data at √s = 7 TeV collected with the ATLAS detector at the LHC during 2011

Measurements of Higgs boson production and couplings in diboson final states with the ATLAS detector at the LHC

Measurements are presented of production properties and couplings of the recently discovered Higgs boson using the decays into boson pairs, H →γ γ, H → Z Z∗ →4l and H →W W∗ →lνlν. The results are based on the complete pp collision data sample recorded by the ATLAS experiment at the CERN Large Hadron Collider at centre-of-mass energies of √s = 7 TeV and √s = 8 TeV, corresponding to an integrated luminosity of about 25 fb−1. Evidence for Higgs boson production through vector-boson fusion is reported. Results of combined fits probing Higgs boson couplings to fermions and bosons, as well as anomalous contributions to loop-induced production and decay modes, are presented. All measurements are consistent with expectations for the Standard Model Higgs boson