AMPA-type glutamate receptors (AMPARs) are responsible for a variety of processes in the mammalian brain including fast excitatory neurotransmission, postsynaptic plasticity, or synapse development. Here, with comprehensive and quantitative proteomic analyses, we demonstrate that native AMPARs are macromolecular complexes with a large molecular diversity. This diversity results from coassembly of the known AMPAR subunits, pore-forming GluA and three types of auxiliary proteins, with 21 additional constituents, mostly secreted proteins or transmembrane proteins of different classes. Their integration at distinct abundance and stability establishes the heteromultimeric architecture of native AMPAR complexes: a defined core with a variable periphery resulting in an apparent molecular mass between 0.6 and 1 MDa. The additional constituents change the gating properties of AMPARs and provide links to the protein dynamics fundamental for the complex role of AMPARs in formation and operation of glutamatergic synapses.
Local Ca 2+ signaling occurring within nanometers of voltage-gated Ca 2+ (Cav) channels is crucial for CNS function, yet the molecular composition of Cav channel nano-environments is largely unresolved. Here, we used a proteomic strategy combining knockoutcontrolled multiepitope affinity purifications with high-resolution quantitative MS for comprehensive analysis of the molecular nano-environments of the Cav2 channel family in the whole rodent brain. The analysis shows that Cav2 channels, composed of poreforming α1 and auxiliary β subunits, are embedded into protein networks that may be assembled from a pool of ∼200 proteins with distinct abundance, stability of assembly, and preference for the three Cav2 subtypes. The majority of these proteins have not previously been linked to Cav channels; about two-thirds are dedicated to the control of intracellular Ca 2+ concentration, including G proteincoupled receptor-mediated signaling, to activity-dependent cytoskeleton remodeling or Ca 2+ -dependent effector systems that comprise a high portion of the priming and release machinery of synaptic vesicles. The identified protein networks reflect the cellular processes that can be initiated by Cav2 channel activity and define the molecular framework for organization and operation of local Ca 2+ signaling by Cav2 channels in the brain.calcium channel | Ca 2+ signaling | proteome | biochemistry | mass spectrometry
SummaryIon fluxes mediated by glial cells are required for several physiological processes such as fluid homeostasis or the maintenance of low extracellular potassium during high neuronal activity. In mice, the disruption of the Cl− channel ClC-2 causes fluid accumulation leading to myelin vacuolation. A similar vacuolation phenotype is detected in humans affected with megalencephalic leukoencephalopathy with subcortical cysts (MLC), a leukodystrophy which is caused by mutations in MLC1 or GLIALCAM. We here identify GlialCAM as a ClC-2 binding partner. GlialCAM and ClC-2 colocalize in Bergmann glia, in astrocyte-astrocyte junctions at astrocytic endfeet around blood vessels, and in myelinated fiber tracts. GlialCAM targets ClC-2 to cell junctions, increases ClC-2 mediated currents, and changes its functional properties. Disease-causing GLIALCAM mutations abolish the targeting of the channel to cell junctions. This work describes the first auxiliary subunit of ClC-2 and suggests that ClC-2 may play a role in the pathology of MLC disease.Video Abstract
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are key modulators of neuronal activity by providing the depolarizing cation current I(h) involved in rhythmogenesis, dendritic integration, and synaptic transmission. These tasks critically depend on the availability of HCN channels, which is dynamically regulated by intracellular cAMP; the range of this regulation, however, largely differs among neurons in the mammalian brain. Using affinity purification and high-resolution mass spectrometry, we identify the PEX5R/Trip8b protein as the beta subunit of HCN channels in the mammalian brain. Coassembly of PEX5R/Trip8b affects HCN channel gating in a subtype-dependent and mode-specific way: activation of HCN2 and HCN4 by cAMP is largely impaired, while gating by phosphoinositides and basal voltage-dependence remain unaffected. De novo expression of PEX5R/Trip8b in cardiomyocytes abolishes beta-adrenergic stimulation of HCN channels. These results demonstrate that PEX5R/Trip8b is an intrinsic auxiliary subunit of brain HCN channels and establish HCN-PEX5R/Trip8b coassembly as a mechanism to control the channels' responsiveness to cyclic nucleotide signaling.
We present a detailed description of the fabrication and operation at room temperature of a novel type of tunnel displacement transducer. Instead of a feedback system it relies on a large reduction factor assuring an inherently stable device. Stability measurements in the tunnel regime infer an electrode stability within 3 pm in a 1 kHz bandwidth. In the contact regime the conductance takes on a discrete number of values when the constriction is reduced atom by atom. This reflects the conduction through discrete channels. © 1995 American Institute of Physics.Micromachining in silicon is an ongoing effort to provide ever smaller devices used as the active part of a sensor. Currently, it is straightforward to produce suspended beams, small springs, and vibrating or rotating structures on a chip. Engineers can make use of a number of classical transducer phenomena, such as piezoelectricity, piezoresistivity and capacitance changes to convert displacements into an electrical signal. However, the formation of smaller sensors is often obtained at the cost of precision, since the signal of the above mentioned transducer phenomena scale with size. In contrast to classical transducers, a tunnel transducer 1 ͑e.g., an STM͒ is compatible with further miniaturization and possesses an astonishing sensitivity to displacements. When a vacuum tunnel gap between two metallic electrodes is increased by 1 Å, the tunnel resistance increases approximately by an order of magnitude. This has been realized by a number of groups who have used tunnel sensors in devices.2 The extreme sensitivity of these sensors on positional displacements however implies that the practical range of operation is limited to distances smaller than 5 Å since at larger distances the resistance becomes almost infinite and unmeasurable.In conventional STM embodiments, one electrode is usually mounted on a flexible lever, which can be moved by an electrical signal. The tunnel gap is kept constant with the use of a feedback system, necessary since temperature fluctuations, ͑acoustic͒ vibrations or other disturbances will otherwise change the vacuum gap over distances much larger than the practical range. An accelerometer, magnetometer, and an infrared sensor have been successfully developed with these kind of tunnel sensors in feedback operation. 2Despite these successes we have used a different approach and constructed an inherently stable tunnel sensor. When used as a displacement sensor this device can be fabricated in such a way that the electrode separation during operation remains in the practical range of about 5 Å. Due to the extreme stability of this device it can be operated without feedback; however it may also be used in a feedback loop. In this letter we present the fabrication and operation of this new type of tunnel sensor which was proposed in Ref. 3. It is inherently stable, adjustable, and compatible with silicon technology. Detailed measurements are shown, in both the contact and tunnel regimes.The principle of operation and a schematic perspec...
Proton extrusion by roots of intact sunflower plants (Helianthus annuas L.) was studied in nutrient solutions or in agar media with a pH indicator. Proton extrusion was enhanced by either iron deficiency, addition of fusicoccin, or single salt solutions of ammonium or potassium salts. The three types of proton extrusion differ in both localization along the roots and capacity. From their sensitivity to ATPase inhibitors it seems justified to characterize them as proton pumps driven by plasma membrane APTases.Enhanced proton extrusion induced by preferential cation uptake from (NH.)2SO4 or K2SO4 was uniformly distributed over the whole root system. In contrast, the enhancement effect of fusicoccin was confied to the basal root zones and that of iron deficiency to the apical root zones. Also the rates of proton extrusion per unit of root fresh weight differed remarkably and increased in the order. Fusicoccin << K2SO4< (NH4hSO4 < iron deficiency.Under iron deficiency the average values of proton extrusion for the whole root system are 5.6 micromoles H' per gram fresh weight per hour, however, for the apical root zones values of about 28 micromoles H can be calculated. This high capacity is most probably related to the iron deficiency-induced formation of rhizodermal transfer cells in the apical root zones. It can be assumed that the various types of root-induced acidification of the rhizosphere are of considerable ecological importance for the plant-soil relationships in general and for mobilization of mineral nutrients from sparingly soluble sources in particular.In higher plants, electrogenic proton pumps are localized in various membranes, such as the plasma membranes, tonoplast or mitochondrial membranes (1-4, 7). It importance. Acidification ofthe soil/root interface (rhizosphere) not only mobilizes mineral nutrients (e.g. P, Fe, Zn; [5,20]) but also affects the activity of microorganisms such as Rhizobium (16) or pathogenic fungi (23,27). This acidification of the rhizosphere can be easily demonstrated by agar techniques and depends on plant species, plant age, form and level of nutrient supply, and buffer capacity of the soil (13). Furthermore, deficiency of phosphorous (8) In this paper we report on results with intact sunflower plants on both the rate of proton extrusion and localization of proton pumps along roots as induced by iron deficiency, FC2 and different nutrient supply. In a following paper the properties of the iron deficiency-induced proton pumps will be described in more detail. MATERIALS AND METHODSGrowth of plants. Seeds of sunflower (Helianthus annuus L. cv Sobrid) were germinated for 3 d in quartz sand moistened with saturated CaS04 solution and subsequently transferred to a continuously aerated nutrient solution (25 plants per 1.5-L plas-
Affinity purification (AP) of protein complexes combined with LC-MS/MS analysis is the current method of choice for identification of protein-protein interactions. Their interpretation with respect to significance, specificity, and selectivity requires quantification methods coping with enrichment factors of more than 1000-fold, variable amounts of total protein, and low abundant, unlabeled samples. We used standardized samples (0.1-1000 fmol) measured on high resolution hybrid linear ion trap instruments (LTQ-FT/Orbitrap) to characterize and improve linearity and dynamic range of label-free approaches. Quantification based on spectral counts was limited by saturation and ion suppression effects with samples exceeding 100 ng of protein, depending on the instrument setup. In contrast, signal intensities of peptides (peak volumes) selected by a novel correlation-based method (TopCorr-PV) were linear over at least 4 orders of magnitude and allowed for accurate relative quantification of standard proteins spiked into a complex protein background. Application of this procedure to APs of the voltage-gated potassium channel Kv1.1 as a model membrane protein complex unambiguously identified the whole set of known interaction partners together with novel candidates. In addition to discriminating these proteins from background, we could determine efficiency, cross-reactivities, and selection biases of the used purification antibodies. The enhanced dynamic range of the developed quantification procedure appears well suited for sensitive identification of specific protein-protein interactions, detection of antibody-related artifacts, and optimization of AP conditions. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.007955, 1-12, 2012.Antibody-based affinity purification (AP) 1 of protein assemblies from biological samples followed by mass spectrometric analysis represents an increasingly popular approach for identification of protein-protein interactions (AP-MS) (1-3). Despite the exquisitely high and specific enrichment theoretically obtainable with antibodies (Abs), this approach faces a number of technical and intrinsic challenges in practice. Target protein complexes typically suffer from poor solubility, instability, and low abundance, particularly when associated with lipid membranes. Moreover, various antibody-related properties such as target selectivity, cross-reactivity, and interference with protein-protein interactions may lead to falsepositive and false-negative results (4). Finally, biological protein-protein interactions may have a more dynamic character, may depend on regulated modifications, or may involve rare protein partners. Together, these effects lead to a significant reduction of AP signal to noise, i.e. low co-enrichment efficiency of interaction partners and significant overlap with background or nonspecific proteins.Classically, AP specificity has been addressed by visualization of purified proteins on one-or two-dimensional gels and comparison of band patterns or spots with those obtained in...
Blue native (BN)1 -PAGE and its colorless variant, colorless native PAGE, were originally developed by Schä gger and co-workers as end point separation methods for characterization of solubilized mitochondrial membrane protein (super-)-complexes under close-to-native conditions (1-3). Subsequently, native gel electrophoresis became the method of choice for first dimension separation followed by second dimension SDS-PAGE in two-dimensional gel-based proteomic analyses (2D-BN) of membrane protein complexes. After staining of the gel-separated proteins, protein spots are individually analyzed by different mass spectrometric methods, and the identified proteins were assigned to complexes based on their co-migration pattern (2D-BN-MS (4)). However, these 2D-BN-MS approaches exhibit the following severe shortcomings: (i) they are critically dependent on the staining properties of individual proteins; (ii) the size resolution of protein complexes is low; and (iii) the assignment of identified proteins to spots and complexes may be ambiguous. Therefore, application of 2D-BN-MS has remained largely restricted to the characterization of highly abundant and well defined membrane protein complexes such as complexes I-V of the respiratory chain in mitochondria (5-7), photosynthetic complexes (8 -10), or viruses (11).In a first attempt to overcome these shortcomings of 2D-BN-MS, Wessels et al. (12) coupled BN-PAGE separation more directly to MS analysis by manually cutting the gel lane into 24 slices/sections of about 2 mm width that were separately digested and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Their study on HEK cell mitochondria identified 59 of the 90 canonical subunits of the oxidative respiratory chain (OXPHOS) complexes I-V. The respective protein abundance profiles (based on standard label-free quantification) showed clustering of their peak maxima into the expected complexes I-V. Since then, this onedimensional BN-MS methodology has been gradually improved with respect to quality of the native gel separation, LC-MS/MS sensitivity, and robustness of the quantitative evaluation. Thus, two recent studies on human mitochondrial preparations (each analyzing two BN separations in 60 and 24 slices, respectively) reported identification and hierarchical profile clustering of 464 (13)
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