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

The neuron-specific K ϩ -Cl Ϫ cotransporter KCC2 extrudes Cl Ϫ and renders GABA and glycine action hyperpolarizing. Thus, it plays a pivotal role in neuronal inhibition. Development-dependent KCC2 activation is regulated at the transcriptional level and by unknown posttranslational mechanisms. Here, we analyzed KCC2 activation at the protein level in the developing rat lateral superior olive (LSO), a prominent auditory brainstem structure. Electrophysiology demonstrated ineffective KCC2-mediated Cl Ϫ extrusion in LSO neurons at postnatal day 3 (P3). Immunohistochemical analyses by confocal and electron microscopy revealed KCC2 signals at the plasma membrane in the somata and dendrites of both immature and mature neurons. Biochemical analysis demonstrated mature glycosylation pattern of KCC2 at both stages. Immunoblot analysis of the immature brainstem demonstrated mainly monomeric KCC2. In contrast, three KCC2 oligomers with molecular masses of ϳ270, ϳ400, and ϳ500 kDa were identified in the mature brainstem. These oligomers were sensitive to sulfhydryl-reducing agents and resistant to SDS, contrary to the situation seen in the related Na ϩ -(K ϩ )-Cl Ϫ cotransporter. In HEK-293 cells, coexpressed hemagglutinin-tagged KCC2 assembled with histidine-tagged KCC2, demonstrating formation of homomers. Based on these findings, we conclude that the oligomers represent KCC2 dimers, trimers, and tetramers. Finally, immunoblot analysis identified a development-dependent increase in the oligomer/monomer ratio from embryonic day 18 to P30 throughout the brain that correlates with KCC2 activation. Together, our data indicate that the developmental shift from depolarization to hyperpolarization can be determined by both increased gene expression and KCC2 oligomerization.

A comprehensive analysis of plasma membrane proteins is essential to in-depth understanding of brain development, function, and diseases. Proteomics offers the potential to perform such a comprehensive analysis, yet it requires efficient protocols for the purification of the plasma membrane compartment. Here, we present a novel and efficient protocol for the separation and enrichment of brain plasma membrane proteins. It lasts only 4 h and is easy to perform. It highly enriches plasma membrane proteins and can be applied to small amounts of brain tissue, such as the cerebellum of a single rat, which was used in the present study. The protocol is based on affinity partitioning of microsomes in an aqueous twophase system. Marker enzyme assays demonstrated a more than 12-fold enrichment of plasma membranes and a strong reduction of other compartments, such as mitochondria and the endoplasmic reticulum. 506 different proteins were identified when the enriched proteins underwent LC-MS/MS analysis subsequent to protein separation by SDS-PAGE. Using gene ontology, 146 proteins were assigned to a subcellular compartment. Ninetythree of those (64%) were membrane proteins, and 49 (34%) were plasma membrane proteins. A combined literature and database search for all 506 identified proteins revealed subcellular information on 472 proteins, of which 197 (42%) were plasma membrane proteins. These comprised numerous transporters, channels, and neurotransmitter receptors, e.g. the inward rectifying potassium channel Kir7.1 and the cerebellum-specific ␥-aminobutyric acid receptor GABRA6. Surface proteins involved in cell-cell contact and disease-related proteins were also identified. Six of the 146 assigned proteins were derived from mitochondrial membranes and 5 from membranes of the endoplasmic reticulum. Taken together, our protocol represents a simple, rapid, and reproducible tool for the proteomic characterization of brain plasma membranes.

Plasma membranes (PMs) are of particular importance for all living cells. They form a selectively permeable barrier to the environment. Many essential tasks of PMs are carried out by their proteinaceous components, including molecular transport, cell-cell interactions, and signal transduction. Due to the key role of these proteins for cellular function, they take center-stage in basic and applied research. A major problem towards in-depth identification and characterization of PM proteins by modern proteomic approaches is their low abundance and immense heterogeneity in different cells. Highly selective and efficient purification protocols are hence essential to any PM proteome analysis. An effective tool for preparative isolation of PMs is partitioning in aqueous polymer two-phase systems. In two-phase systems, membranes are separated according to differences in surface properties rather than size and density. Despite their rare application to the fractionation of animal tissues and cells, they represent an attractive alternative to conventional fractionation protocols. Here, we review the principles of partitioning using aqueous polymer two-phase systems and compare aqueous polymer two-phase systems with other methods currently used for the isolation of PMs.

In the majority of neurons, the intracellular Cl− concentration is set by the activity of the Na+‐K+‐2Cl− cotransporter (NKCC1) and the K+‐Cl− cotransporter (KCC2). Here, we investigated the cotransporters’ functional dependence on membrane rafts. In the mature rat brain, NKCC1 was mainly insoluble in Brij 58 and co‐distributed with the membrane raft marker flotillin‐1 in sucrose density flotation experiments. In contrast, KCC2 was found in the insoluble fraction as well as in the soluble fraction, where it co‐distributed with the non‐raft marker transferrin receptor. Both KCC2 populations displayed a mature glycosylation pattern. Disrupting membrane rafts with methyl‐β‐cyclodextrin (MβCD) increased the solubility of KCC2, yet had no effect on NKCC1. In human embryonic kidney‐293 cells, KCC2 was strongly activated by a combined treatment with MβCD and sphingomyelinase, while NKCC1 was inhibited. These data indicate that membrane rafts render KCC2 inactive and NKCC1 active. In agreement with this, inactive KCC2 of the perinatal rat brainstem largely partitioned into membrane rafts. In addition, the exposure of the transporters to MβCD and sphingomyelinase showed that the two transporters differentially interact with the membrane rafts. Taken together, membrane raft association appears to represent a mechanism for co‐ordinated regulation of chloride transporter function.

Comprehensive knowledge about the plasma membrane protein profile of a given brain region, at defined developmental stages, will greatly foster the understanding of brain function and dysfunction. Protocols are required which selectively enrich plasma membranes from small brain regions, thereby resulting in high yields. Here, we present a suitable protocol that is based on aqueous polymer two-phase systems. It is material saving, easy to perform, fast, and low-priced. Evidence for its effectiveness was obtained by marker enzyme assays, immunoblot analyses, and mass spectrometry. Plasma membranes from all parts of the cells (somata, dendrites, and axons) were enriched, whereas there was a reduction of mitochondria and endoplasmic reticulum. The total of 15.0% of the initial activity of the plasma membrane marker was recovered, while the activity of the mitochondrial marker and the marker for the endoplasmic reticulum was 0.2% of the initial activity. Mass spectrometric analyses of proteins purified from approximately one-fourth of rat cerebellum (i.e., 80 mg of tissue) resulted in the identification of 525 different proteins, with 27.3% (gene ontology) or 38.2% (gene cards) being allocated to the plasma membrane. When accepting 4.7% of the initial mitochondrial marker activity and 2.9% of the initial activity of the marker for the endoplasmic reticulum as contaminations, the yield of the plasma membrane marker increased to 28.8%. Under these conditions, 586 different proteins were identified by mass spectrometry, 26.1-36.5% of which were plasma membrane proteins. Taken together, our protocol represents a powerful tool for the analysis of the plasma membrane subproteome of distinct brain regions.

The brain is unquestionably the most fascinating organ. Despite tremendous progress, current knowledge falls short of being able to explain its function. An emerging approach toward improved understanding of the molecular mechanisms underlying brain function is neuroproteomics. Today's neuroscientists have access to a battery of versatile technologies both in transcriptomics and proteomics. The challenge is to choose the right strategy in order to generate new hypotheses on how the brain works. The goal of this review is therefore two-fold: first we recall the bewildering cellular, molecular, and functional complexity in the brain, as this knowledge is fundamental to any study design. In fact, an impressive complexity on the molecular level has recently re-emerged as a central theme in large-scale analyses. Then we review transcriptomics and proteomics technologies, as both are complementary. Finally, we comment on the most widely used proteomics techniques and their respective strengths and drawbacks. We conclude that for the time being, neuroproteomics should focus on its strengths, namely the identification of posttranslational modifications and protein-protein interactions, as well as the characterization of highly purified subproteomes. For global expression profiling, emphasis should be put on further development to significantly increase coverage.

Subcellular fractionation represents an essential technique for functional proteome analysis. Recently, we provided a subcellular fractionation protocol for minute amounts of tissue that yielded a nuclear fraction, a membrane and organelle fraction, and a cytosolic fraction. In the current study, we attempted to improve the protocol for the isolation of integral membrane proteins, as these are particularly important for brain function. In the membrane and organelle fraction, we increased the yield of membranes and organelles by about 50% by introducing a single re-extraction step. We then tested two protocols towards their capacity to enrich membrane proteins present in the membrane and organelle fraction. One protocol is based on sequential solubilization using subsequent increases of chaotropic conditions such as urea, thereby partitioning hydrophobic proteins from hydrophilic ones. The alternative protocol applies high-salt and high-pH washes to remove non-membrane proteins. The enrichment of membrane proteins by these procedures, as compared to the original membrane and organelle fraction, was evaluated by 16-BAC-SDS-PAGE followed by mass spectrometry of randomly selected spots. In the original membrane and organelle fraction, 7 of 50 (14%) identified proteins represented integral membrane proteins, and 15 (30%) were peripheral membrane proteins. In the urea-soluble fraction, 4 of 33 (12%) identified proteins represented integral membrane proteins, and 10 (30%) were peripheral membrane proteins. In the high-salt/high-pH resistant sediment, 12 of 45 (27%) identified proteins were integral membrane proteins and 13 (29%) represented peripheral membrane proteins. During the analysis, several proteins involved in neuroexocytosis were detected, including syntaxin, NSF, and Rab3-interaction protein 2. Taken together, differential centrifugation in combination with high-salt and high-pH washes resulted in the highest enrichment of integral membrane proteins and, therefore, represents an adequate technique for region-specific profiling of membrane proteins in the brain.