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...