Blue Native PAGE Research Articles

Novel Indomethacin Action: Selective and Direct Activation of Protein Kinase C-ε

Background/Aims: In our earlier study, indomethacin potentiated α7 acetylcholine (ACh) receptor responses by activating protein kinase C (PKC). The present study was conducted to gain further insight into the indomethacin action on PKC. Methods: PKC activity was assayed in PC-12 cells or under the cell-free conditions. PKC-ε was knocked-down using the siRNA to silence the PKC-ε-targeted gene. A fluorescein-conjugated indomethacin was synthesized to examine the interaction of indomethacin with PKC-ε. Results: In the in situ PKC assay, indomethacin activated PKC in PC-12 cells in a concentration (1-100 µM)-dependent manner, and the activation was suppressed by knocking-down PKC-ε. In the cell-free PKC assay, indomethacin (100 µM) activated PKC-ε in the absence of diacylglycerol, phosphatidylserine, and calcium, but other PKC isozymes such as α, βΙ, βΙΙ, γ, δ, ι, and ζ were not activated. In the indomethacin binding assay using a fluorescent-conjugated indomethacin on blue native-polyacrylamide gel electrophoresis (blue native-PAGE), a fluorescent signal was detected at the site consistent with PKC-ε protein and the signal was attenuated by adding non-conjugated indomethacin or eliminated by pretreatment with non-conjugated indomethacin. Conclusion: The results of the present study show that indomethacin has the potential to selectively activate PKC-ε through its direct binding, independently of cyclooxygenase (COX) inhibition.

FLU, a negative feedback regulator of tetrapyrrole biosynthesis, is physically linked to the final steps of the Mg++-branch of this pathway

Regulation of tetrapyrrole biosynthesis in higher plants has been attributed to negative feedback control. Two effectors of feedback inhibition have been identified, heme and the FLU protein. Inhibition by heme implicates the Fe-branch via regulation of the initial step of tetrapyrrole synthesis. In the present work a FLU-containing chloroplast membrane complex was identified, that besides FLU comprises the four enzymes catalyzing the final steps of chlorophyll synthesis. The results support the notion that FLU links chlorophyll synthesis and the target of feedback control, glutamyl-tRNA reductase, thereby allowing also the Mg-branch to control the initial step of tetrapyrrole synthesis. Structured summary of protein interactionsFLU, CHL27, PORB, PORC and CHLPphysically interact by blue native page (View interaction)FLUphysically interacts with CHL27, PORB, PORC, CHLP and GluTR by anti tag co-immunoprecipitation (View interaction)FLUphysically interacts with CHL27 by anti bait co-immunoprecipitation (View Interaction: 1, 2)FLUphysically interacts with CHL27 by anti tag co-immunoprecipitation (View interaction)FLUphysically interacts with CHL27, PORB, PORC and CHLP by anti tag co-immunoprecipitation (View interaction)

The cytosolic domain of human Tom22 modulates human Bax mitochondrial translocation and conformation in yeast

The role of the mitochondrial protein receptor Tom22p in the interaction of pro-apoptotic protein Bax with yeast mitochondria was investigated. Co-immunoprecipitation assays showed that human Bax interacted with different TOM subunits, including Tom22p. Expression of the cytosolic receptor domain of human Tom22 increased Bax mitochondrial localization, but decreased the proportion of active Bax. BN–PAGE showed that the cytosolic domain of Tom22 interfered with the oligomerization of Bax. These data suggest that the interaction with the cytosolic domain of Tom22 helps Bax to acquire a conformation able to interact with the outer mitochondrial membrane. Structured summary of protein interactionsBAX and BAXphysically interact with TOM22 by anti bait coimmunoprecipitation (View interaction)BAXphysically interacts with TOM70 by anti bait coimmunoprecipitation (View interaction)BAXphysically interacts with TOM22, TOM70 and TOM40 by anti bait coimmunoprecipitation (View interaction)BAXphysically interacts with TOM22 by anti bait coimmunoprecipitation (View interaction)BAX and BAXbind by blue native page (View interaction)BAXphysically interacts with TOM40 by anti bait coimmunoprecipitation (View interaction)

OEP80, an essential protein paralogous to the chloroplast protein translocation channel Toc75, exists as a 70-kD protein in the Arabidopsis thaliana chloroplast outer envelope

Toc75 and OEP80 are paralogous proteins found in the Viridiplantae lineages, and appear to have evolved from a protein in the outer membrane of an ancient cyanobacterium. Toc75 is known to act as a protein translocation channel at the outer membrane of the chloroplast envelope, whereas the exact function of OEP80 is not understood. In Arabidopsis thaliana, each protein is encoded by a single gene, and both are essential for plant viability from embryonic stages onward. Sequence annotation and immunoblotting data with an antibody against its internal sequence (αOEP80(325-337)) indicated that the molecular weight of OEP80 is ca. 80kD. Here we present multiple data to show that the size of A. thaliana OEP80 is smaller than previously estimated. First, we prepared the antibody against a recombinant protein consisting of annotated full-length A. thaliana OEP80 with an N-terminal hexahistidine tag (αOEP80(1-732)). This antibody recognized a 70-kD protein in the A. thaliana chloroplast membrane fraction which migrated faster than the His-tagged antigen and the protein recognized by the αOEP80(325-337) antibody on SDS-PAGE. Immunoprecipitation followed by LC-MS/MS analysis confirmed that the 70-kD protein was encoded by the OEP80 cDNA. Next, we performed a genetic complementation assay using embryo-lethal oep80-null plants and constructs encoding OEP80 and its variants. The results revealed that the nucleotide sequence encoding the 52 N-terminal amino acids was not required for functional expression of OEP80 and accumulation of the 70-kD protein. The data also indicated that an additional C-terminal T7 tag remained intact without disrupting the functionality of OEP80, and was not exposed to the cytoplasmic surface of the chloroplast envelope. Finally, OEP80-T7 and Toc75 showed distinct migration patterns on blue native-PAGE. This study provides molecular tools to investigate the function of OEP80, and also calls for caution in using an anti-peptide antibody.

Biochemical Characterization of Human ZIP13 Protein: A HOMO-DIMERIZED ZINC TRANSPORTER INVOLVED IN THE SPONDYLOCHEIRO DYSPLASTIC EHLERS-DANLOS SYNDROME

The human SLC39A13 gene encodes ZIP13, a member of the LZT (LIV-1 subfamily of ZIP zinc transporters) family. The ZIP13 protein is important for connective tissue development, and its loss of function is causative for the spondylocheiro dysplastic form of Ehlers-Danlos syndrome. However, this protein has not been characterized in detail. Here we report the first detailed biochemical characterization of the human ZIP13 protein using its ectopic expressed and the purified recombinant protein. Protease accessibility, microscopic, and computational analyses demonstrated that ZIP13 contains eight putative transmembrane domains and a unique hydrophilic region and that it resides with both its N and C termini facing the luminal side on the Golgi. Analyses including cross-linking, immunoprecipitation, Blue Native-PAGE, and size-exclusion chromatography experiments indicated that the ZIP13 protein may form a homo-dimer. We also demonstrated that ZIP13 mediates zinc influx, as assessed by monitoring the expression of the metallothionein gene and by detecting the intracellular zinc level with a zinc indicator, FluoZin-3. Our data indicate that ZIP13 is a homo-dimerized zinc transporter that possesses some domains that are not found in other LZT family members. This is the first biochemical characterization of the physiologically important protein ZIP13 and the demonstration of homo-dimerization for a mammalian ZIP zinc transporter family member. This biochemical characterization of the human ZIP13 protein provides important information for further investigations of its structural characteristics and function.

Identifying transient protein–protein interactions in EphB2 signaling by blue native PAGE and mass spectrometry

Receptor tyrosine kinases (RTKs) are proteins that upon ligand stimulation undergo dimerization and autophosphorylation. Eph receptors (EphRs) are RTKs that are found in different cell types, from both tissues that are developing and from mature tissues, and play important roles in the development of the central nervous system and peripheral nervous system. EphRs also play roles in synapse formation, neural crest formation, angiogenesis and in remodeling the vascular system. Interaction of EphRs with their ephrin ligands lead to activation of signal transduction pathways and formation of many transient protein-protein interactions that ultimately leads to cytoskeletal remodeling. However, the sequence of events at the molecular level is not well understood. We used blue native PAGE and MS to analyze the transient protein-protein interactions that resulted from the stimulation of EphB2 receptors by their ephrinB1-Fc ligands. We analyzed the phosphotyrosine-containing protein complexes immunoprecipitated from the cell lysates of both unstimulated (-) and ephrinB1-Fc-stimulated (+) NG108 cells. Our experiments allowed us to identify many signaling proteins, either known to be part of EphB2 signaling or new for this pathway, which are involved in transient protein-protein interactions upon ephrinB1-Fc stimulation. These data led us to investigate the roles of proteins such as FAK, WAVEs and Nischarin in EphB2 signaling.

Coordination of carbon fixation and nitrogen metabolism in Salicornia europaea under salinity: Comparative proteomic analysis on chloroplast proteins

Halophyte, like Salicornia europaea, could make full use of marginal saline land for carbon fixation. How the photosynthesis of S. europaea is regulated under high salinity implicates a significant aspect to exploit this pioneer plant in future. Measurement of photosynthesis parameters demonstrated the reduction of photosynthesis for the 0 and 800 mM NaCl treated plants are more likely due to non-stomatal limitation, which might be caused by changes in the enzymes associated with photosynthesis. Different salinity induced ultrastructure changes other than photosynthetic apparatus damage, suggesting the photosynthesis of S. europaea might be affected via biochemical regulation. Comparative proteomics analysis of chloroplast proteins by 2-D gel electrophoresis reproducibly detected 90 differentially expressed proteins, among which 66 proteins were identified by nanoLC MS/MS. Further study of thylakoid membrane proteins by Blue-Native PAGE proved the increase in abundance of light reaction proteins under salinity. Analysis of gene expression patterns of 12 selected proteins provides evidence for the correlations between transcription and proteomics data. Based on our results, a putative model of photosynthesis regulatory network figured out proper coordination of carbon fixation and nitrogen metabolism in chloroplast of S. europaea under salinity, which provided subcellular level insight into salt tolerance mechanism in S. europaea.

Translocators YopB and YopD from Yersinia enterocolitica Form a Multimeric Integral Membrane Complex in Eukaryotic Cell Membranes

The type III secretion systems are contact-activated secretion systems that allow bacteria to inject effector proteins across eukaryotic cell membranes. The secretion apparatus, called injectisome or needle complex, includes a needle that terminates with a tip structure. The injectisome exports its own distal components, like the needle subunit and the needle tip. Upon contact, it exports two hydrophobic proteins called translocators (YopB and YopD in Yersinia enterocolitica) and the effectors. The translocators, assisted by the needle tip, form a pore in the target cell membrane, but the structure of this pore remains elusive. Here, we purified the membranes from infected sheep erythrocytes, and we show that they contain integrated and not simply adherent YopB and YopD. In blue native PAGE, these proteins appeared as a multimeric 500- to 700-kDa complex. This heteropolymeric YopBD complex could be copurified after solubilization in 0.5% dodecyl maltoside but not visualized in the electron microscope. We speculate that this complex may not be stable and rigid but only transient.

Chaperonin TRiC assists the refolding of sperm-specific glyceraldehyde-3-phosphate dehydrogenase

The cytosolic chaperonin TRiC was isolated from ovine testes using ultracentrifugation and heparin-Sepharose chromatography. The molecular mass of the obtained preparation was shown to exceed 900 kDa (by Blue Native PAGE). SDS–PAGE yielded a set of bands in the range of 50–60 kDa. Electron microscopy examination revealed ring-shaped complexes with the outer diameter of 15 nm and the inner diameter of approximately 6 nm. The results suggest that the purified chaperonin is an oligomeric complex composed of two 8-membered rings. The chaperonin TRiC was shown to assist an ATP-dependent refolding of recombinant forms of sperm-specific glyceraldehyde-3-phosphate dehydrogenase, an enzyme that is expressed only in precursor cells of the sperms in the seminiferous tubules of the testes. In contrast, TRiC did not influence the refolding of muscle isoform of glyceraldehyde-3-phosphate dehydrogenase and assisted the refolding of muscle lactate dehydrogenase by an ATP-independent mechanism. The obtained results suggest that TRiC is likely to be involved in the refolding of sperm-specific proteins.

Structure and function of the glucose PTS transporter from Escherichia coli

The glucose transporter IICB of the Escherichia coli phosphotransferase system (PTS) consists of a polytopic membrane domain (IIC) responsible for substrate transport and a hydrophilic C-terminal domain (IIB) responsible for substrate phosphorylation. We have overexpressed and purified a triple mutant of IIC (mut-IIC), which had recently been shown to be suitable for crystallization purposes. Mut-IIC was homodimeric as determined by blue native-PAGE and gel-filtration, and had an eyeglasses-like structure as shown by negative-stain transmission electron microscopy (TEM) and single particle analysis. Glucose binding and transport by mut-IIC, mut-IICB and wildtype-IICB were compared with scintillation proximity and in vivo transport assays. Binding was reduced and transport was impaired by the triple mutation. The scintillation proximity assay allowed determination of substrate binding, affinity and specificity of wildtype-IICB by a direct method. 2D crystallization of mut-IIC yielded highly-ordered tubular crystals and made possible the calculation of a projection structure at 12Å resolution by negative-stain TEM. Immunogold labeling TEM revealed the sidedness of the tubular crystals, and high-resolution atomic force microscopy the surface structure of mut-IIC. This work presents the structure of a glucose PTS transporter at the highest resolution achieved so far and sets the basis for future structural studies.

Isolation and purification of the major photosynthetic antenna, fucoxanthin-Chl a/c protein, from cultured discoid germilings of the brown Alga, Cladosiphon okamuranus TOKIDA (Okinawa Mozuku).

A chlorophyll c binding membrane intrinsic light-harvesting complex, the fucoxanthin-chlorophyll a/c protein (FCP), was isolated from cultured discoid germilings of an edible Japanese brown alga, Cladosiphon (C.) okamuranus TOKIDA (Okinawa Mozuku in Japanese). The discoid germiling is an ideal source of brown algal photosynthetic pigment-protein complexes in terms of its size and easiness of cultivation on a large scale. Ion-exchange chromatography was crucial for the purification of FCP from solubilized thylakoid proteins. The molecular weight of the purified FCP assembly was estimated to be ~56kDa using blue native-PAGE. Further subunit analyses using 2D-PAGE revealed that the FCP assembled as a trimer consisting of two distinguishable subunits having molecular weights of 18.2 (H) and 17.5 (L)kDa. Fluorescence and fluorescence-excitation spectra confirmed that the purified FCP assembly was functionally intact.

The orexin OX1 receptor exists predominantly as a homodimer in the basal state: potential regulation of receptor organization by both agonist and antagonist ligands

It is unclear what proportion of a G-protein-coupled receptor is present in cells as dimers or oligomers. Saturation bioluminescence resonance energy transfer studies demonstrated the orexin OX(1) receptor to be present in such complexes. Forms of this receptor containing a minimal epitope tag, with the C-terminus linked to yellow fluorescent protein or modified at the N-terminus to incorporate a SNAP tag, migrated in SDS/PAGE gels as monomers, indicating a lack of covalent interactions. Solubilization with dodecylmaltoside, followed by Blue native-PAGE, indicated that the receptor constructs migrated predominantly as anticipated for dimeric species with evidence for further, higher-order, complexes, and this was true over a wide range of expression levels. Addition of SDS prior to separation by Blue native-PAGE resulted in much of the previously dimeric, and all of the higher-order, complexes being dissociated and now migrating at the size predicted for monomeric species. Expression of forms of the OX(1) receptor capable of generating enzyme complementation confirmed that solubilization itself did not result in interaction artefacts. Addition of the endogenous agonist orexin A enhanced the proportion of higher-order OX(1) receptor complexes, whereas selective OX(1) antagonists increased the proportion the OX(1) receptor migrating in Blue native-PAGE as a monomer. The antagonist effects were produced in a concentration-dependent manner, consistent with the affinity of the ligands for the receptor. Homogeneous time-resolved fluorescence resonance energy transfer studies using Tag-Lite™ reagents on cells expressing the SNAP-tagged OX(1) receptor identified cell-surface OX(1) homomers. Predominantly at low receptor expression levels, orexin A increased such fluorescence resonance energy transfer signals, also consistent with ligand-induced reorganization of the homomeric complex.

Complex Formation, Phosphorylation, and Localization of Protein Kinase A ofSchizosaccharomyces pombeupon Glucose Starvation

Nine sam mutants that undergo sexual differentiation without requiring starvation in Schizosaccharomyces pombe were previously isolated. In this study, we identified a nonsense mutation on the pka1 locus in the sam6 mutant. pka1 encodes a catalytic subunit of protein kinase A (PKA). Replacement and overexpression of pka1 suppressed the KCl sensitivity and hyper-mating phenotype of sam6, confirming that sam6 is an allele of pka1. To characterize further the regulation of Pka1, we tested the physical interaction between Pka1 and Cgs1 (a regulatory subunit of PKA). Pka1 and Cgs1 physically interacted under glucose-limited conditions but not under glucose-rich conditions. In addition, the formation of a Pka1-Cgs1 complex was detected under glucose-limited conditions by Blue Native PAGE. Furthermore, the Pka1 protein was found to be phosphorylated under glucose-starved conditions, and at the same time its localization shifted from the nucleus towards the cytoplasm (mainly the vacuoles), suggesting a strong relationship among phosphorylation, complex formation, and the cytoplasmic distribution of Pka1.

Insights into the Composition and Assembly of the Membrane Arm of Plant Complex I through Analysis of Subcomplexes in Arabidopsis Mutant Lines

NADH-ubiquinone oxidoreductase (Complex I, EC 1.6.5.3) is the largest complex of the mitochondrial respiratory chain. In eukaryotes, it is composed of more than 40 subunits that are encoded by both the nuclear and mitochondrial genomes. Plant Complex I differs from the enzyme described in other eukaryotes, most notably due to the large number of plant-specific subunits in the membrane arm of the complex. The elucidation of the assembly pathway of Complex I has been a long-standing research aim in cellular biochemistry. We report the study of Arabidopsis mutants in Complex I subunits using a combination of Blue-Native PAGE and immunodetection to identify stable subcomplexes containing Complex I components, along with mass spectrometry analysis of Complex I components in membrane fractions and two-dimensional diagonal Tricine SDS-PAGE to study the composition of the largest subcomplex. Four subcomplexes of the membrane arm of Complex I with apparent molecular masses of 200, 400, 450, and 650 kDa were observed. We propose a working model for the assembly of the membrane arm of Complex I in plants and assign putative roles during the assembly process for two of the subunits studied.

Functional Stability of Unliganded Envelope Glycoprotein Spikes among Isolates of Human Immunodeficiency Virus Type 1 (HIV-1)

The HIV-1 envelope glycoprotein (Env) spike is challenging to study at the molecular level, due in part to its genetic variability, structural heterogeneity and lability. However, the extent of lability in Env function, particularly for primary isolates across clades, has not been explored. Here, we probe stability of function for variant Envs of a range of isolates from chronic and acute infection, and from clades A, B and C, all on a constant virus backbone. Stability is elucidated in terms of the sensitivity of isolate infectivity to destabilizing conditions. A heat-gradient assay was used to determine T90 values, the temperature at which HIV-1 infectivity is decreased by 90% in 1 h, which ranged between ∼40 to 49°C (n = 34). For select Envs (n = 10), the half-lives of infectivity decay at 37°C were also determined and these correlated significantly with the T90 (p = 0.029), though two ‘outliers’ were identified. Specificity in functional Env stability was also evident. For example, Env variant HIV-1ADA was found to be labile to heat, 37°C decay, and guanidinium hydrochloride but not to urea or extremes of pH, when compared to its thermostable counterpart, HIV-1JR-CSF. Blue native PAGE analyses revealed that Env-dependent viral inactivation preceded complete dissociation of Env trimers. The viral membrane and membrane-proximal external region (MPER) of gp41 were also shown to be important for maintaining trimer stability at physiological temperature. Overall, our results indicate that primary HIV-1 Envs can have diverse sensitivities to functional inactivation in vitro, including at physiological temperature, and suggest that parameters of functional Env stability may be helpful in the study and optimization of native Env mimetics and vaccines.

Monitoring cytoplasmic protein complexes with blue native gel electrophoresis and stable isotope labelling with amino acids in cell culture: Analysis of changes in the 20S proteasome

Analysis of protein complexes is of increasing interest in the field of proteomics. A challenge is to develop methods for monitoring changes in the quantity and subunit composition of protein complexes on a proteome-wide scale. Here, we describe the combination of 1-D blue native polyacrylamide gel electrophoresis (BN-PAGE) with stable isotope labelling of amino acids in cell culture (SILAC) and tandem mass spectrometry (MS/MS). Cleared lysates from normal and perturbed samples, one incorporating heavy stable isotopes and the other light isotopes, are co-separated by blue native PAGE and then analysed and quantitated with MS/MS and appropriate software. This permits the analysis of cytoplasmic complexes. To demonstrate this technique, we explored how the 20S proteasome changes when the Pre9/α3 subunit, the only non-essential subunit of this complex, was deleted. Our results showed that ΔPre9/α3 cells can form the 20S proteasome complex, although with reduced efficiency. This involves an increase in expression of the α4 subunit. Our findings suggest this technique as an approach for the study of quantitative and qualitative differences in protein complexes, from cleared cell lysates.

Design and synthesis of new fluorescent probe for rapid and highly sensitive detection of proteins via electrophoretic gel stain

A new fluorescent molecular probe, 2,2'-(1E,1'E)-2,2'-(4-(dicyanomethylene)-4H-pyrane-2,6-diyl)bis(ethene-2,1-diyl)bis(sodium benzenesulfonate) salt (1), possessing the cyanopyranyl moieties and two benzene sulfonic acid groups was designed and synthesized to detect proteins in solution and for high-throughput SDS-PAGE. Compound 1 exhibited no fluorescence in the absence of proteins; however, it exhibited strong fluorescence on the addition of bovine serum albumin as a result of intramolecular charge transfer. Compared with the conventional protocols for in-gel protein staining, such as SYPRO Ruby and silver staining, 1 achieves higher sensitivity, even though it offers a simplified, higher throughput protocol. In fact, the total time required for protein staining was 60-90 min under optimum conditions much shorter than that required by the less-sensitive silver staining or SYPRO Ruby staining protocols. Moreover, 1 was successfully applied to protein identification by mass spectrometry via in-gel tryptic digestion, Western blotting, and native PAGE together with protein staining by 1, which is a modified protocol of blue native PAGE (BN-PAGE). Thus, 1 may facilitate high-sensitivity protein detection, and it may be widely applicable as a convenient tool in various scientific and medical fields.

Homodimerization of the Src Homology 3 Domain of the Calcium Channel β-Subunit Drives Dynamin-dependent Endocytosis

Voltage-dependent calcium channels constitute the main entry pathway for calcium into excitable cells. They are heteromultimers formed by an α(1) pore-forming subunit (Ca(V)α(1)) and accessory subunits. To achieve a precise coordination of calcium signals, the expression and activity of these channels is tightly controlled. The accessory β-subunit (Ca(V)β), a membrane associated guanylate kinase containing one guanylate kinase (β-GK) and one Src homology 3 (β-SH3) domain, has antagonistic effects on calcium currents by regulating different aspects of channel function. Although β-GK binds to a conserved site within the α(1)-pore-forming subunit and facilitates channel opening, β-SH3 binds to dynamin and promotes endocytosis. Here, we investigated the molecular switch underlying the functional duality of this modular protein. We show that β-SH3 homodimerizes through a single disulfide bond. Substitution of the only cysteine residue abolishes dimerization and impairs internalization of L-type Ca(V)1.2 channels expressed in Xenopus oocytes while preserving dynamin binding. Covalent linkage of the β-SH3 dimerization-deficient mutant yields a concatamer that binds to dynamin and restores endocytosis. Moreover, using FRET analysis, we show in living cells that Ca(V)β form oligomers and that this interaction is reduced by Ca(V)α(1). Association of Ca(V)β with a polypeptide encoding the binding motif in Ca(V)α(1) inhibited endocytosis. Together, these findings reveal that β-SH3 dimerization is crucial for endocytosis and suggest that channel activation and internalization are two mutually exclusive functions of Ca(V)β. We propose that a change in the oligomeric state of Ca(V)β is the functional switch between channel activator and channel internalizer.

All functional aquaporin-4 isoforms are expressed in the rat cochlea and contribute to the formation of orthogonal arrays of particles

The water channel aquaporin-4 (AQP4) is expressed in the cochlea and is essential for normal hearing. Unlike other AQPs, multiple isoforms of AQP4 have been reported in diverse tissues, three of which, M1, M23, and Mz, function as water channels. In addition, these protein isoforms are found in higher order complexes. Morphologically these higher order complexes correspond to orthogonal arrays of particles (OAPs) that are found in cell membranes by freeze fracture analysis. Using RT-PCR, quantitative PCR and blue-native PAGE immunoblots we identified all functional AQP4 isoforms -M1, M23, and Mz- and the formation of higher-order complexes in the organ of Corti of the rat. Complementary freeze-fracture studies revealed OAPs distributed in the lateral and basal membrane domains of the cochlear duct supporting cells, specifically Hensen's cells and outer sulcus cells. The unique inter- and intracellular heterogeneity in size, density and shape of OAPs suggests exceptional physiological requirements for the maintenance of water homeostasis during auditory sensory transduction in the cochlea.

Mass spectrometric analysis of GABAAreceptor subtypes and phosphorylations from mouse hippocampus

The brain GABA(A) receptor (GABA(A) R) is a key element of signaling and neural transmission in health and disease. Recently, complete sequence analysis of the recombinant GABA(A) R has been reported, separation and mass spectrometrical (MS) characterisation from tissue, however, has not been published so far. Hippocampi were homogenised, put on a sucrose gradient 10-69% and the layer from 10 to 20% was used for extraction of membrane proteins by a solution of Triton X-100, 1.5 M aminocaproic acid in the presence of 0.3 M Bis-Tris. This mixture was subsequently loaded onto blue native PAGE (BN-PAGE) with subsequent analysis on denaturing gel systems. Spots from the 3-DE electrophoretic run were stained with Colloidal Coomassie Brilliant Blue, and spots with an apparent molecular weight between 40 and 60 kDa were picked and in-gel digested with trypsin, chymotrypsin and subtilisin. The resulting peptides were analysed by nano-LC-ESI-MS/MS (ion trap) and protein identification was carried out using MASCOT searches. In addition, known GABA(A) R-specific MS information taken from own previous studies was used for searches of GABA(A) R subunits. β-1, β-2 and β-3, θ and ρ-1 subunits were detected and six novel phosphorylation sites were observed and verified by phosphatase treatment. The method used herein enables identification of several GABA(A) R subunits from mouse hippocampus along with phosphorylations of β-1 (T227, Y230), β-2 (Y215, T439) and β-3 (T282, S406) subunits. The procedure forms the basis for GABA(A) R studies at the protein chemical rather than at the immunochemical level in health and disease.