Jens Schindler

Oligomerization of KCC2 correlates with development of inhibitory neurotransmission

The neuron-specific K+–Cl− cotransporter KCC2 extrudes Cl− 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− 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 ∼270, ∼400, and ∼500 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+–(K+)–Cl− 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.

Proteomic Analysis of Brain Plasma Membranes Isolated by Affinity Two-phase Partitioning

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 two-phase 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. Ninety-three 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 γ-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. Because it conserves membrane structure and protein interactions, it is also suitable to enrich multimeric protein complexes from the plasma membrane for subsequent analysis.

Opposite effect of membrane raft perturbation on transport activity of KCC2 and NKCC1

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.

Enrichment of integral membrane proteins from small amounts of brain tissue

Summary.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.

Two-Dimensional Separation of Membrane Proteins by 16-BAC-SDS-PAGE

Defining membrane proteomes is fundamental to our understanding of many physiological and pathophysiological processes. Their separation and identification is hence a key issue in basic and biomedical research. Due to their hydrophobic character, few high-resolution techniques for separation are available for qualitative and quantitative approaches. For gel-based methods, the two-dimensional 16-BAC/SDS-PAGE is the method of choice. This technique separates proteins in the first dimension using an acidic buffer system and the cationic detergent benzyldimethyl-n-hexadecylammonium chloride (16-BAC) and in the second dimension by SDS-PAGE. Here, we describe a detailed protocol for the separation of proteins by 16-BAC/SDS-PAGE.

Monitoring the native phosphorylation state of plasma membrane proteins from a single mouse cerebellum

Neuronal processing in the cerebellum involves the phosphorylation and dephosphorylation of various plasma membrane proteins such as AMPA or NMDA receptors. Despite the importance of changes in phosphorylation pattern, no global phospho-proteome analysis has yet been performed. As plasma membrane proteins are major targets of the signalling cascades, we developed a protocol to monitor their phosphorylation state starting from a single mouse cerebellum. An aqueous polymer two-phase system was used to enrich for plasma membrane proteins. Subsequently, calcium phosphate precipitation, immobilized metal affinity chromatography, and TiO(2) were combined to a sequential extraction procedure prior to mass spectrometric analyses. This strategy resulted in the identification of 1501 different native phosphorylation sites in 507 different proteins. 765 (51%) of these phosphorylation sites were localized with a confidence level of 99% or higher. 41.4% of the identified proteins were allocated to the plasma membrane and about half of the phosphorylation sites have not been reported previously. A bioinformatic screen for 12 consensus sequences identified putative kinases for 642 phosphorylation sites. In summary, the protocol deployed here identified several hundred novel phosphorylation sites of cerebellar proteins. Furthermore, it provides a valuable tool to monitor the plasma membrane proteome from any small brain samples of interest under differing physiological or pathophysiological conditions.

Enrichment of Brain Plasma Membranes by Affinity Two-Phase Partitioning

Plasma membranes encompass a complex and varying set of proteins essential to life. In addition, plasma membrane proteins represent the majority of all known drug targets. The characterization of plasma membrane proteomes is, therefore, of eminent importance. A current bottleneck is the lack of efficient protocols to isolate plasma membranes from tissues or entire organs. To this end, we recently established a simple and effective isolation procedure which is based on aqueous polymer two-phase systems. In this chapter, we provide a detailed protocol for the isolation of plasma membranes from brain tissue, which could easily be adapted to other sources.

Isolation of Plasma Membranes from the Nervous System by Countercurrent Distribution in Aqueous Polymer Two-Phase Systems

The plasma membrane separates the cell-interior from the cells environment. To maintain homeostatic conditions and to enable transfer of information, the plasma membrane is equipped with a variety of different proteins such as transporters, channels, and receptors. The kind and number of plasma membrane proteins are a characteristic of each cell type. Owing to their location, plasma membrane proteins also represent a plethora of drug targets. Their importance has entailed many studies aiming at their proteomic identification and characterization. Therefore, protocols are required that enable their purification in high purity and quantity. Here, we report a protocol, based on aqueous polymer two-phase systems, which fulfils these demands. Furthermore, the protocol is time-saving and protects protein structure and function.