Polycystin-2, the product of the gene mutated in type 2 autosomal dominant polycystic kidney disease (ADPKD), is the prototypical member of a subfamily of the transient receptor potential (TRP) channel superfamily, which is expressed abundantly in the endoplasmic reticulum (ER) membrane. Here, we show by single channel studies that polycystin-2 behaves as a calcium-activated, high conductance ER channel that is permeable to divalent cations. Epithelial cells overexpressing polycystin-2 show markedly augmented intracellular calcium release signals that are lost after carboxy-terminal truncation or by the introduction of a disease-causing missense mutation. These data suggest that polycystin-2 functions as a calcium-activated intracellular calcium release channel in vivo and that polycystic kidney disease results from the loss of a regulated intracellular calcium release signalling mechanism.

Estrogen receptors (ERs) are believed to be ligand-activated transcription factors belonging to the nuclear receptor superfamily, which on ligand binding translocate into the nucleus and activate gene transcription. To date, two ERs have been identified: ER␣ and ER␤. ER␣ plays major role in the estrogen-mediated genomic actions in both reproductive and nonreproductive tissue, whereas the function of ER␤ is still unclear. In this study, we used immunocytochemistry, immunoblotting, and proteomics to demonstrate that ER␤ localizes to the mitochondria. In immunocytochemistry studies, ER␤ was detected with two ER␤ antibodies and found to colocalize almost exclusively with a mitochondrial marker in rat primary neuron, primary cardiomyocyte, and a murine hippocampal cell line. The colocalization of ER␤ and mitochondrial markers was identified by both fluorescence and confocal microscopy. No translocation of ER␤ into the nucleus on 17␤-estradiol treatment was seen by using immunocytochemistry. Immunoblotting of purified human heart mitochondria showed an intense signal of ER␤, whereas no signals for nuclear and other organelle markers were found. Finally, purified human heart mitochondrial proteins were separated by SDS͞PAGE. The 50,000 -65,000 Mr band was digested with trypsin and subjected to matrix-assisted laser desorption͞ionization mass spectrometric analysis, which revealed seven tryptic fragments that matched with those of ER␤. In summary, this study demonstrated that ER␤ is localized to mitochondria, suggesting a role for mitochondrial ER␤ in estrogen effects on this important organelle.nuclear receptor ͉ mitochondria E strogens play an important role in development, growth, and differentiation of both female and male secondary sex characteristics. Estrogen receptors (ERs) were the first identified nuclear receptor family member (1). The first ER, now called ER␣, was cloned in 1986 (2, 3). A second ER␤, was identified and cloned a decade later (4, 5). Like other members of the nuclear receptor superfamily, both ERs have a modular structure consisting of distinct functional domains (1). The DNA-binding domain (DBD) enables the receptor to bind its cognate target site consisting of an inverted repeat of two half-sites with the consensus motif AG-GTCA spaced by 3 bp, referred to as an estrogen response element (ERE). The ligand-binding domain enables estrogen binding to the receptors. ERs are highly conserved between ER␣ and ER␤, with Ͼ95% homology for the DBD and Ϸ50% homology for the ligand-binding domain. Less homology is observed for the transactivational domain between ER␣ and ER␤ (5, 6).Genomic actions of ER␣ are well described (7). On binding to ER␣, estrogens induce a conformational change in the ER␣ proteins, which is accompanied by the dissociation of the accessory protein, heat shock protein 90, thereby exposing the DBD. In the nucleus, the receptor-ligand complex binds to DNA and modulates gene transcription. This transcriptional͞translational activation is comparatively slow and sensitive to cyclohexi...

Molecular cloning has introduced an unexpected diversity of neurotransmitter receptors. In this study we review the types, the localization and possible synaptic function of the inhibitory neurotransmitter receptors in the mammalian retina. Glycine receptors (GlyRs) and their localization in the mammalian retina were analyzed immunocytochemically. Specific antibodies against the alpha 1 subunit of the GlyR (mAb2b) and against all subunits of the GlyR (mAb4a) were used. Both antibodies produced a punctate immunofluorescence, which was shown by electron microscopy to represent clustering of GlyRs at synaptic sites. Synapses expressing the alpha 1 subunit of the GlyR were found on ganglion cell dendrites and on bipolar cell axons. GlyRs were also investigated in the oscillator mutant mouse. The complete loss of the alpha 1 subunit was compensated for by an apparent upregulation of the other subunits of the GlyR. GABAA receptors (GABAARs) and their retinal distribution were studied with specific antibodies that recognize the alpha 1, alpha 2, alpha 3, beta 1, beta 2, beta 3, gamma 2 and delta subunits. Most antibodies produced a punctate immunofluorescence in the inner plexiform layer (IPL) which was shown by electron microscopy to represent synaptic clustering of GABAARs. The density of puncta varied across the IPL and different subunits were found in characteristic strata. This stratification pattern was analyzed with respect to the ramification of cholinergic amacrine cells. Using intracellular injection with Lucifer yellow followed by immunofluorescence, we found that GABAARs composed of different subunits were expressed by the same ganglion cell, however, they were clustered at different synaptic sites. The distribution of GABAC receptors was studied in the mouse and in the rabbit retina using an antiserum that recognizes the rho 1, rho 2 and rho 3 subunits. GABAC receptors were found to be clustered at postsynaptic sites. Most, if not all of the synapses were found on rod and cone bipolar axon terminals. In conclusion we find a great diversity of glycine and GABA receptors in the mammalian retina, which might match the plethora of morphological types of amacrine cells. This may also point to subtle differences in synaptic function still to be elucidated.

Nuclear calcium (Ca 2؉ ) regulates a number of important cellular processes, including gene transcription, growth, and apoptosis. However, it is unclear whether Ca 2؉ signaling is regulated differently in the nucleus and cytosol. To investigate this possibility, we examined subcellular mechanisms of Ca 2؉ release in the HepG2 liver cell line. The type II isoform of the inositol 1,4,5-trisphosphate (InsP 3) receptor (InsP 3R) was expressed to a similar extent in the endoplasmic reticulum and nucleus, whereas the type III InsP 3R was concentrated in the endoplasmic reticulum, and the type I isoform was not expressed. Ca 2؉ signals induced by low InsP3 concentrations started earlier or were larger in the nucleus than in the cytosol, indicating higher sensitivity of nuclear Ca 2؉ stores for InsP3. Nuclear InsP3R channels were active at lower InsP 3 concentrations than InsP3R from cytosol. Enriched expression of type II InsP 3R in the nucleus results in greater sensitivity of the nucleus to InsP 3, thus providing a mechanism for independent regulation of Ca 2؉ -dependent processes in this cellular compartment.

Synapse-associated proteins are the scaffold for the selective aggregation of ion channels at synapses; they provide the link to cytoskeletal elements and possibly are involved with the regulation of synaptic efficacy by electrical activity. The localization of the postsynaptic density protein PSD-95 was studied in different mammalian retinae (rat, monkey, and tree shrew) by using immunocytochemical methods. Immunofluorescence for PSD-95 was most prominent in the outer plexiform layer (OPL). The axon terminals of rods and cones, the rod spherules and cone pedicles, were strongly labeled. Electron microscopy, using preembedding immunocytochemistry, showed PSD-95 localized presynaptically within the photoreceptor terminals. Distinct PSD-95 labeling was also present in the inner plexiform layer (IPL). It had a punctate appearance suggesting the synaptic clustering of PSD-95 in the IPL. Electron microscopy showed that PSD-95 was concentrated in processes that were postsynaptic at bipolar cell ribbon synapses (dyads). As a rule, only one of the two postsynaptic members of the dyad was labeled for PSD-95. Double-labeling experiments were performed for PSD-95 and for SAP 102 or PSD-93, respectively, two other members of the family of synapse-associated proteins. All three were found to be colocalized in the synaptic hot spots in the IPL. In the OPL, however, PSD-95 and PSD-93 were found presynaptically, whereas SAP 102 was located postsynaptically at photoreceptor synapses. Double-labeling experiments also were performed for PSD-95 and for the NR1 subunit of the NMDA receptor. They were found to be colocalized in synaptic hot spots in the IPL. Key words: PSD-95; SAP 102; PSD-93; NMDA receptors; NR1 subunit; postsynaptic density; receptor clustering; cone pedicle; rod spheruleThe postsynaptic density (PSD) is a complex network of transmitter receptors, anchoring proteins, and cytoskeletal elements (Kennedy, 1997). The selective concentration of transmitter receptors in PSDs not only is essential for the reliable and rapid synaptic transmission but also plays an important role in synaptic plasticity and possibly in memory formation (Mammen et al., 1997;Rao and Craig, 1997;K irsch and Betz, 1998).The postsynaptic density protein PSD-95 belongs to a family of ion channel clustering molecules (for review, see Gomperts, 1996;Sheng, 1996;Kennedy, 1997). In its N-terminal part PSD-95 contains three highly homologous regions of ϳ90 amino acids. They have been called PDZ domains because they have been found in postsynaptic density proteins (PSDs), in the Drosophila tumor suppressor gene "disks large" (Dlg), and in cell -cell adhesion proteins like zonula occludens-1 (Z O1). In mammals this family of postsynaptic density proteins includes four closely related proteins: PSD-95/SAP 90 (Cho et al., 1992;K istner et al., 1993), SAP 97/ hdlg (L ue et al., 1994;Müller et al., 1995), PSD-93/Chapsyn-110 (Brenman et al., 1996a;K im et al., 1996), and SAP 102 (Müller et al., 1996).The second PDZ domain in PSD-95 has been shown by the yeast...

The synaptic localization of the kainate receptor subunits GluR6/7 and KA2 and of the ionotropic glutamate receptor subunits delta1/2 was studied in the rat retina using receptor-specific antisera. GluR6/7 and KA2 were present in both synaptic layers of the retina: the inner plexiform layer (IPL) and the outer plexiform layer (OPL). The localization of delta1/2 was restricted to the IPL. Detailed ultrastructural examination showed that in the OPL GluR6/7 was localized in horizontal cell processes postsynaptic to both rod spherules and cone pedicles. It was always only one of the two invaginating horizontal cell processes at the photoreceptor synapses labeled for GluR6/7. KA2 in the OPL was found only postsynaptic to cone pedicles and never postsynaptic to rod spherules. The KA2-labeled processes made flat contacts with the cone pedicles, suggesting they are the dendrites of OFF bipolar cells. In the IPL the different receptor subunits were localized postsynaptically to ribbon synapses of both rod and cone bipolar cells. As a rule, only one of the two postsynaptic elements at the bipolar cell dyad was stained for each of the receptor subunits examined. The selective and heterogeneous distribution of these receptors at the ribbon synapses of the OPL and IPL suggests a high degree of differential processing of the glutamatergic signals.

Although neurofibrillary tangle (NFT) formation is a central event in both familial and sporadic Alzheimer's disease (AD), neither cellular origin nor functional consequence of the NFTs are fully understood. This largely is due to the lack of available in vivo models for neurofibrillary degeneration (NFD). NFTs have only been identified in transgenic mice, bearing a transgene for a rare hereditary neurodegenerative disease, frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP17). Epidemiological evidence suggests a much higher occurrence of dementia in stroke patients. This may represent the underlying cause of the pathogenesis of sporadic AD, which accounts for the majority of AD cases. We examined pathological markers of AD in a rodent stroke model. Here we show that after transient cerebral ischemia, hyperphosphorylated tau accumulates in neurons of the cerebral cortex in the ischemic area, forms filaments similar to those present in human neurodegenerative tauopathies and colocalizes with markers of apoptosis. As a potential underlying mechanism, we were able to determine that transient ischemia induced tau hyperphosphorylation and NFT-like conformations are associated with aberrant activation of cyclin dependent kinase 5 (Cdk5) and can be rescued by delivery of a potent, but non-specific cyclin dependent kinase inhibitor, roscovitine to the brain. Our study further indicates that accumulation of p35 and its calpain-mediated cleavage product, p25 may account for the deregulation of Cdk5 induced by transient ischemia. We conclude that Cdk5 may be the principal protein kinase responsible for tau hyperphosphorylation and may be a hallmark of the tauopathies in this stroke model.

Polyclonal antibodies which recognize the rho-subunits of the GABA(C) receptor were applied to sections of the rat retina. Strong punctate immunoreactivity was found in the inner plexiform layer (IPL), which was shown by electron microscopy to represent a clustering of the GABA(C) receptors at synaptic sites. During postnatal development diffuse rho-immunoreactivity was first observed at postnatal day P3. Distinct labelling of bipolar cells appeared at P7 and punctate, synaptic labelling was observed at P10. In order to show that the rho-immunoreactive puncta coincide with the axons of bipolar cells, double immunostainings of retinal sections with an antiserum against syntaxin 3 and with the rho-antiserum were performed. The experiments showed that rho-immunoreactive puncta are preferentially located on the axon terminals of rod and cone bipolar cells. In order to determine whether GABA(C) receptor rho-subunits coassemble with GABA(A) receptor subunits, double-labelling experiments were performed with subunit specific antisera. Punctate, putative synaptic clustering was observed with all antisera applied, however, GABA(C) receptor expressing puncta did not coincide with GABA(A) receptor containing puncta. This suggests that there are no synaptic GABA receptors in the retina in which GABA(A) and GABA(C) receptor subunits are coassembled. Similar double-labelling experiments were also performed to find out whether GABA(C) receptors and glycine receptors are colocalized. They were clustered at different synapses. This suggests that synaptic GABA(C) receptors consist of rho-subunits and are not coassembled with GABA(A)- or glycine-receptor subunits.