A plasma membrane Ca2+ ATPase isoform at the postsynaptic density.

Most excitatory input in the hippocampus impinges on dendritic spines. Entry of Ca(2+) into spines through NMDA receptors can trigger a sequence of biochemical reactions leading to sustained changes in synaptic efficacy. To provide specificity, dendritic spines restrict the diffusion of Ca(2+) signaling and downstream molecules. The postsynaptic density (PSD) (the most prominent subdomain within the spine) is the site of Ca(2+) entry through NMDA receptors. We here demonstrate that Ca(2+) can also be removed via pumps embedded in the PSD. Using light- and electron-microscopic immunohistochemistry, we find that PMCA2w, a member of the plasma membrane Ca(2+)-ATPase (PMCA) family, concentrates at the PSD of most hippocampal spines. We propose that PMCA2w may be recruited into supramolecular complexes at the postsynaptic density, thus helping to regulate Ca(2+) nanodomains at subsynaptic sites. Taken together, these results suggest a novel function for PMCAs as modulators of Ca(2+) signaling at the synapse.

Molecular interactions of the plasma membrane calcium ATPase 2 at pre- and post-synaptic sites in rat cerebellum.

The plasma membrane calcium extrusion mechanism, PMCA (plasma membrane calcium ATPase) isoform 2 is richly expressed in the brain and particularly the cerebellum. Whilst PMCA2 is known to interact with a variety of proteins to participate in important signalling events [Strehler EE, Filoteo AG, Penniston JT, Caride AJ (2007) Plasma-membrane Ca(2+) pumps: structural diversity as the basis for functional versatility. Biochem Soc Trans 35 (Pt 5):919-922], its molecular interactions in brain synapse tissue are not well understood. An initial proteomics screen and a biochemical fractionation approach identified PMCA2 and potential partners at both pre- and post-synaptic sites in synapse-enriched brain tissue from rat. Reciprocal immunoprecipitation and GST pull-down approaches confirmed that PMCA2 interacts with the post-synaptic proteins PSD95 and the NMDA glutamate receptor subunits NR1 and NR2a, via its C-terminal PDZ (PSD95/Dlg/ZO-1) binding domain. Since PSD95 is a well-known partner for the NMDA receptor this raises the exciting possibility that all three interactions occur within the same post-synaptic signalling complex. At the pre-synapse, where PMCA2 was present in the pre-synapse web, reciprocal immunoprecipitation and GST pull-down approaches identified the pre-synaptic membrane protein syntaxin-1A, a member of the SNARE complex, as a potential partner for PMCA2. Both PSD95-PMCA2 and syntaxin-1A-PMCA2 interactions were also detected in the molecular and granule cell layers of rat cerebellar sagittal slices by immunohistochemistry. These specific molecular interactions at cerebellar synapses may allow PMCA2 to closely control local calcium dynamics as part of pre- and post-synaptic signalling complexes.

Plasma-membrane Ca(2+) pumps: structural diversity as the basis for functional versatility.

Plasma-membrane calcium pumps [PMCAs (plasma-membrane Ca(2+)-ATPases)] expel Ca(2+) from eukaryotic cells to maintain overall Ca(2+) homoeostasis and to provide local control of intracellular Ca(2+) signalling. Recent work indicates functional versatility among PMCA isoforms, with specific pumps being essential for cochlear hair cell function, sperm motility, feedback signalling in the heart and pre- and post-synaptic Ca(2+) regulation in neurons. The functional versatility of PMCAs is due to differences in their regulation by CaM (calmodulin), kinases and other signalling proteins, as well as to their differential targeting and retention in defined plasma membrane domains. The basis for this is the structural diversity of PMCAs. In mammals, four genes encode PMCA isoforms 1-4, and each of these has multiple variants generated by alternative RNA splicing. The alternatively spliced regions are intimately involved in the regulatory interactions and differential membrane localization of the pumps. The alternatively spliced C-terminal tail acts as an autoinhibitory domain by interacting with the catalytic core of the pump. The degree of inhibition and the kinetics of interaction with the major activator CaM differ between PMCA variants. This translates into functional differences in how PMCAs handle Ca(2+) signals of different magnitude and frequency. Accumulating evidence thus demonstrates how structural diversity provides functional versatility in the PMCAs.

Human calmodulin-like protein is an epithelial-specific protein regulated during keratinocyte differentiation.

Human calmodulin-like protein (CLP) is a calcium-binding protein down-regulated in a cell culture model of mammary tumorigenesis as well as in a majority of breast cancers in vivo. CLP down-regulation may be a result of the poorly differentiated state of these cell lines and tumors, or CLP expression may be incompatible with the uncontrolled cell growth associated with tumorigenesis. To learn more about CLP expression and regulation, we determined the distribution of CLP in various human tissues by immunohistochemistry. CLP was expressed exclusively in the epithelium of the tissues surveyed and was most abundant in thyroid, breast, prostate, kidney, and skin. CLP expression appears to increase in stratified epithelium during differentiation, as illustrated in the skin where CLP staining intensified from the basal through the spinous to the granular layers. Using a normal human keratinocyte culture model, we examined CLP expression in response to various agents known to affect keratinocyte differentiation. Agents that inhibit (epidermal growth factor, EGF) or permit (keratinocyte growth factor) terminal differentiation correspondingly regulate CLP expression. Factors modulating the EGF receptor signaling pathway were particularly potent in regulating CLP expression. CLP expression correlated with an agent's ability to promote terminal differentiation regardless of the agent's effect on keratinocyte proliferation. These studies show that CLP expression is coordinately regulated by, and may be involved in, the program of terminal differentiation in human keratinocytes and, likely, other differentiating epithelial cell types.

Plasma membrane calcium ATPase plays a role in reducing Ca(2+)-mediated cytotoxicity in PC12 cells.

In many cell types, cell death induced by a variety of insults is accompanied by an increase in intracellular calcium. The Ca(2+) homeostatic mechanisms affected by such insults, however, have not been fully determined. Recent evidence indicates that kainic acid-induced seizures alter plasma membrane calcium ATPase mRNA expression within vulnerable hippocampal cell populations before the onset of cell death. We examined the effects of altering plasma membrane calcium ATPase expression on cell vulnerability in rat pheochromocytoma 12 cells. Pheochromocytoma 12 cells are vulnerable to Ca(2+) overload induced by the Ca(2+) ionophore A23187. Reverse transcriptase-PCR and Western blot data indicated that plasma membrane calcium ATPase isoform 4b constitutes a major calcium pump isoform in the pheochromocytoma 12 cells. Therefore, permanently transfected pheochromocytoma 12-derived cell lines were established that either over-expressed plasma membrane calcium ATPase isoform 4b, or suppressed the expression of the endogenous plasma membrane calcium ATPase isoform 4. Over-expressing clones were less vulnerable to Ca(2+)-mediated cell death induced by A23187 whereas "antisense" clones were considerably more susceptible. These data indicate that regulation of plasma membrane calcium ATPase expression may be critical to cellular survival when cells are exposed to pathological increases in intracellular calcium.

The tumor-sensitive calmodulin-like protein is a specific light chain of human unconventional myosin X.

Human calmodulin-like protein (CLP) is an epithelial-specific Ca(2+)-binding protein whose expression is strongly down-regulated in cancers. Like calmodulin, CLP is thought to regulate cellular processes via Ca(2+)-dependent interactions with specific target proteins. Using gel overlays, we identified a approximately 210-kDa protein binding specifically and in a Ca(2+)-dependent manner to CLP, but not to calmodulin. Yeast two-hybrid screening yielded a CLP-interacting clone encoding the three light chain binding IQ motifs of human "unconventional" myosin X. Pull-down experiments showed CLP binding to the IQ domain to be direct and Ca(2+)-dependent. CLP interacted strongly with IQ motif 3 (K(d) approximately 0.5 nm) as determined by surface plasmon resonance. Epitope-tagged myosin X was localized preferentially at the cell periphery in MCF-7 cells, and CLP colocalized with myosin X in these cells. Myosin X was able to coprecipitate CLP and, to a lesser extent, calmodulin from transfected COS-1 cells, indicating that CLP is a specific light chain of myosin X in vivo. Because unconventional myosins participate in cellular processes ranging from membrane trafficking to signaling and cell motility, myosin X is an attractive CLP target. Altered myosin X regulation in (tumor) cells lacking CLP may have as yet unknown consequences for cell growth and differentiation.

Plasma membrane Ca2+-atpase isoforms 2b and 4b interact promiscuously and selectively with members of the membrane-associated guanylate kinase family of PDZ (PSD95/Dlg/ZO-1) domain-containing proteins.

Spatial and temporal regulation of intracellular Ca(2+) signaling depends on localized Ca(2+) microdomains containing the requisite molecular components for Ca(2+) influx, efflux, and signal transmission. Plasma membrane Ca(2+)-ATPase (PMCA) isoforms of the "b" splice type contain predicted PDZ (PSD95/Dlg/ZO-1) interaction domains. The COOH-terminal tail of PMCA2b isolated the membrane-associated guanylate kinase (MAGUK) protein SAP97/hDlg as a binding partner in a yeast two-hybrid screen. The related MAGUKs SAP90/PSD95, PSD93/chapsyn-110, SAP97, and SAP102 all bound to the COOH-terminal tail of PMCA4b, whereas only the first three bound to the tail of PMCA2b. Coimmunoprecipitations confirmed the interaction selectivity between PMCA4b and SAP102 as opposed to the promiscuity of PMCA2b and 4b in interacting with other SAPs. Confocal immunofluorescence microscopy revealed the exclusive presence and colocalization of PMCA4b and SAP97 in the basolateral membrane of polarized Madin-Darby canine kidney epithelial cells. In hippocampal neurons, PMCA2b was abundant throughout the somatodendritic compartment and often extended into the neck and head of individual spines where it colocalized with SAP90/PSD95. These data show that PMCA "b" splice forms interact promiscuously but also with specificity with different members of the PSD95 family of SAPs. PMCA-SAP interactions may play a role in the recruitment and maintenance of the PMCA at specific membrane domains involved in local Ca(2+) regulation.

Differentiation induces up-regulation of plasma membrane Ca(2+)-ATPase and concomitant increase in Ca(2+) efflux in human neuroblastoma cell line IMR-32.

Precise regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is achieved by the coordinated function of Ca(2+) channels and Ca(2+) buffers. Neuronal differentiation induces up-regulation of Ca(2+) channels. However, little is known about the effects of differentiation on the expression of the plasma membrane Ca(2+)-ATPase (PMCA), the principal Ca(2+) extrusion mechanism in neurons. In this study, we examined the regulation of PMCA expression during differentiation of the human neuroblastoma cell line IMR-32. [Ca(2+)](i) was monitored in single cells using indo-1 microfluorimetry. When the Ca(2+)-ATPase of the endoplasmic reticulum was blocked by cyclopiazonic acid, [Ca(2+)](i) recovery after small depolarization-induced Ca(2+) loads was governed primarily by PMCAs. [Ca(2+)](i) returned to baseline by a process described by a monoexponential function in undifferentiated cells (tau = 52 +/- 4 s; n = 25). After differentiation for 12-16 days, the [Ca(2+)](i) recovery rate increased by more than threefold (tau = 17 +/- 1 s; n = 31). Western blots showed a pronounced increase in expression of three major PMCA isoforms in IMR-32 cells during differentiation, including PMCA2, PMCA3 and PMCA4. These results demonstrate up-regulation of PMCAs on the functional and protein level during neuronal differentiation in vitro. Parallel amplification of Ca(2+) influx and efflux pathways may enable differentiated neurons to precisely localize Ca(2+) signals in time and space.

Role of alternative splicing in generating isoform diversity among plasma membrane calcium pumps.

Calcium pumps of the plasma membrane (also known as plasma membrane Ca(2+)-ATPases or PMCAs) are responsible for the expulsion of Ca(2+) from the cytosol of all eukaryotic cells. Together with Na(+)/Ca(2+) exchangers, they are the major plasma membrane transport system responsible for the long-term regulation of the resting intracellular Ca(2+) concentration. Like the Ca(2+) pumps of the sarco/endoplasmic reticulum (SERCAs), which pump Ca(2+) from the cytosol into the endoplasmic reticulum, the PMCAs belong to the family of P-type primary ion transport ATPases characterized by the formation of an aspartyl phosphate intermediate during the reaction cycle. Mammalian PMCAs are encoded by four separate genes, and additional isoform variants are generated via alternative RNA splicing of the primary gene transcripts. The expression of different PMCA isoforms and splice variants is regulated in a developmental, tissue- and cell type-specific manner, suggesting that these pumps are functionally adapted to the physiological needs of particular cells and tissues. PMCAs 1 and 4 are found in virtually all tissues in the adult, whereas PMCAs 2 and 3 are primarily expressed in excitable cells of the nervous system and muscles. During mouse embryonic development, PMCA1 is ubiquitously detected from the earliest time points, and all isoforms show spatially overlapping but distinct expression patterns with dynamic temporal changes occurring during late fetal development. Alternative splicing affects two major locations in the plasma membrane Ca(2+) pump protein: the first intracellular loop and the COOH-terminal tail. These two regions correspond to major regulatory domains of the pumps. In the first cytosolic loop, the affected region is embedded between a putative G protein binding sequence and the site of phospholipid sensitivity, and in the COOH-terminal tail, splicing affects pump regulation by calmodulin, phosphorylation, and differential interaction with PDZ domain-containing anchoring and signaling proteins. Recent evidence demonstrating differential distribution, dynamic regulation of expression, and major functional differences between alternative splice variants suggests that these transporters play a more dynamic role than hitherto assumed in the spatial and temporal control of Ca(2+) signaling. The identification of mice carrying PMCA mutations that lead to diseases such as hearing loss and ataxia, as well as the corresponding phenotypes of genetically engineered PMCA "knockout" mice further support the concept of specific, nonredundant roles for each Ca(2+) pump isoform in cellular Ca(2+) regulation.

The promoter region of the human PMCA1 gene mediates transcriptional downregulation by 1,25-dihydroxyvitamin D(3).

The gene for plasma membrane calcium pump isoform1 (PMCA1) is expressed in calcium-transporting epithelia and bone mesenchymal cells and is upregulated to 1,25-(OH)(2)D(3) in those tissues. A candidate sequence for a vitamin D response element (VDRE) is present within a 1.7-kb promoter region of the human PMCA1 (hPMCA1) gene. We studied hPMCA1 promoter activity in MDBK and ROS 17/2.8 cell lines as PMCA1 mRNA expression is upregulated by 1,25-(OH)(2)D(3) in both. Structural analysis of the putative hPMCA1 VDRE sequence was performed using mobility shift analysis (EMSA) and nuclear extracts from COS-1 cells expressing human VDR (hVDR) and RXRalpha (hRXRalpha). 1,25-(OH)(2)D(3) induced transrepression of the entire 1.7-kb hPMCA1 promoter and of one promoter deletion construct in ROS 17/2.8 cells but not MDBK cells when assayed by luciferase reporter gene assays. Three additional hPMCA1 promoter deletion constructs were unaffected by 1,25-(OH)(2)D(3) in either cell line. While hVDR and hRXRalpha were capable of complexing with a rat osteocalcin DR3 VDRE, EMSA analysis of the potential VDRE from the hPMCA1 gene did not show interaction of either nuclear receptor. Our results indicate tissue-specific sensitivity of the promoter region of the hPMCA1 gene to direct transcriptional downregulation by 1,25-(OH)(2)D(3) and suggest that any positive regulatory VDRE must lie outside of the 1.7-kb core promoter.

The calmodulin multigene family as a unique case of genetic redundancy: multiple levels of regulation to provide spatial and temporal control of calmodulin pools?

Calmodulin (CaM) is a ubiquitous, highly conserved calcium sensor protein involved in the regulation of a wide variety of cellular events. In vertebrates, an identical CaM protein is encoded by a family of non-allelic genes, raising questions concerning the evolutionary pressure responsible for the maintenance of this apparently redundant family. Here we review the evidence that the control of the spatial and temporal availability of CaM may require multiple regulatory levels to ensure the proper localization, maintenance and size of intracellular CaM pools. Differential transcription of the CaM genes provides one level of regulation to meet tissue-specific, developmental and cell-specific needs for altered CaM levels. Post-transcriptional regulation occurs at the level of mRNA stability, perhaps dependent on alternative polyadenylation and differences in the untranslated sequences of the multiple gene transcripts. Recent evidence indicates that trafficking of specific CaM mRNAs may occur to specialized cellular locales such as the dendrites of neurons. This could allow local CaM synthesis and thereby help generate local pools of CaM. Local CaM activity may be further regulated by post-translational mechanisms such as phosphorylation or storage of CaM in a 'masked' form. The spatial resolution of CaM activity is enhanced by the limited free diffusion of CaM combined with differential affinity for and availability of target proteins. Preserving multiple CaM genes with divergent noncoding sequences may be necessary in complex organisms to ensure that the many CaM-dependent processes occur with the requisite spatial and temporal resolution. Transgenic mouse models and studies on mice carrying single and double gene 'knockouts' promise to shed further light on the role of specificity versus redundancy in the evolutionary maintenance of the vertebrate CaM multigene family.

Plasma membrane calcium ATPases as critical regulators of calcium homeostasis during neuronal cell function.

The plasma membrane calcium ATPases (PMCAs) are ubiquitously expressed proteins that couple the extrusion of calcium across the plasma membrane with the hydrolysis of ATP. In mammals, four separate genes encode distinct PMCA isoforms. Complex patterns of alternative RNA splicing generate additional isoform variability. Functionally, the PMCAs were originally assigned the role of maintaining basal levels of intracellular calcium. Recent evidence, however, is expanding the role of the PMCAs as important participants in dynamic Ca2+ regulation and as crucial players of Ca2+ export during normal and pathological conditions. This review highlights recent advances made on the biology of the PMCAs within the context of neuronal development, cellular responses to external stimuli and cell survival. Particular emphasis is placed on the role of the PMCAs in vestibular and auditory functions, localized calcium signaling in photoreceptor synaptic terminals and calcium-mediated cell death.

Loss of immunoreactivity for human calmodulin-like protein is an early event in breast cancer development.

Cell proliferation requires calmodulin, a protein that regulates calcium-dependent enzymes involved in signal transduction pathways in eukaryotic cells. Calmodulin-like protein (CLP) is found in certain epithelial cell types, including normal breast epithelium, and, although it closely resembles calmodulin in amino acid sequence, CLP interacts with different proteins than does calmodulin. The observation that CLP mRNA expression is dramatically reduced in transformed breast epithelial cells led to two hypotheses: (1) CLP helps to maintain the differentiated state in epithelial cells; and (2) downregulation of CLP accompanies malignant transformation of breast epithelial cells. The objective of this study was to determine if the expression of CLP in human breast cancer specimens is reduced in comparison to its expression in normal breast tissue. Eighty human breast cancer biopsy specimens were analyzed immunohistochemically for CLP expression by using a polyclonal rabbit antihuman CLP antibody. CLP expression was reduced in 79% to 88% of the invasive ductal carcinoma and lobular carcinoma specimens and in a similar fraction of the ductal carcinoma in-situ specimens, compared with normal breast specimens. None of the breast cancer specimens showed an increase in CLP expression. These findings support the hypotheses that CLP behaves as a functional tumor suppressor protein and is downregulated early in breast cancer progression.

Sequential assignment of 1H, 15N, 13C resonances and secondary structure of human calmodulin-like protein determined by NMR spectroscopy.

Human calmodulin-like protein (CLP) is closely related to vertebrate calmodulin, yet its unique cell specific expression pattern, overlapping but divergent biochemical properties, and specific target proteins suggest that it is not an isoform of calmodulin. To gain insight into the structural differences that may underlie the difference target specificities and biochemical properties of CLP when compared to calmodulin, we determined the sequential backbone assignment and associated secondary structure of 144 out of the 148 residues of Ca2+-CLP by using multinuclear multidimensional NMR spectroscopy. Despite a very high overall degree of structural similarity between CLP and calmodulin, a number of significant differences were found mainly in the length of alpha-helices and in the central nonhelical flexible region. Interestingly, the regions of greatest primary sequence divergence between CLP and calmodulin in helices III and VIII displayed only minor secondary structure differences. The data suggest that the distinct differences in target specificity and biochemical properties of CLP and calmodulin result from the sum of several minor structural and side-chain changes spread over multiple domains in these proteins.

Characterization of the human CALM2 calmodulin gene and comparison of the transcriptional activity of CALM1, CALM2 and CALM3.

Human calmodulin is encoded by three genes CALM1, CALM2 and CALM3 located on different chromosomes. To complete the characterization of this family, the exon-intron structure of CALM2 was solved by a combination of genomic DNA library screening and genomic PCR amplification. Intron interruptions were found at identical positions in human CALM2 as in CALM1 and CALM3; however, the overall size of CALM2 (16 kb) was almost twice that of the other two human CALM genes. Over 1 kb of the 5' flanking sequence of human CALM2 were determined, revealing the presence of a TATA-like sequence 27 nucleotides upstream of the transcriptional start site and several conserved sequence elements possibly involved in the regulation of this gene. To determine if differential transcriptional activity plays a major role in regulating cellular calmodulin levels, we directly measured and compared the mRNA abundance and transcriptional activity of the three CALM genes in proliferating human teratoma cells. CALM3 was at least 5-fold more actively transcribed than CALM1 or CALM2. CALM transcriptional activity agreed well with the mRNA abundance profile in the teratoma cells. In transient transfections using luciferase reporter genes driven by 1 kb of the 5' flanking DNA of the three CALM genes, the promoter activity correlated with the endogenous CALM transcriptional activity, but only when the 5' untranslated regions were included in the constructs. We conclude that the CALM gene family is differentially active at the transcriptional level in teratoma cells and that the 5' untranslated regions are necessary to recover full promoter activation.

Minimum CAG repeat in the human calmodulin-1 gene 5' untranslated region is required for full expression.

The human calmodulin-1 gene (hCALM1) contains a (CAG)7 repeat in its 5'-untranslated region (5'-UTR). We found this repeat to be stable and nonpolymorphic in the human population. To determine whether the repeat region affects hCALM1 expression and whether repeat expansions to numbers known to be associated with disease in other genes may alter expression, we tested luciferase reporter genes driven by the hCALM1 promoter and 5'-UTR containing 0, 7 (wild-type), 20, and 45 CAG repeats in human NT2/D1 teratoma cells. Interestingly, the repeat deletion, (CAG)0, decreased expression by 45%, while repeat expansions to (CAG)20 and (CAG)45, or the insertion of a scrambled (C,A,G)7 sequence did not alter gene expression. These data indicate (1) that the endogenous repeat element is required for full expression of hCALM1, and (2) that some triplet repeat expansions in the 5'-UTR of protein-coding genes may be well tolerated and even optimize gene expression.

Plasma membrane Ca2+ ATPase isoform 4b binds to membrane-associated guanylate kinase (MAGUK) proteins via their PDZ (PSD-95/Dlg/ZO-1) domains.

Plasma membrane Ca2+ ATPases are P-type pumps important for intracellular Ca2+ homeostasis. The extreme C termini of alternatively spliced "b"-type Ca2+ pump isoforms resemble those of K+ channels and N-methyl-D-aspartate receptor subunits that interact with channel-clustering proteins of the membrane-associated guanylate kinase (MAGUK) family via PDZ domains. Yeast two-hybrid assays demonstrated strong interaction of Ca2+ pump 4b with the PDZ1 + 2 domains of several mammalian MAGUKs. Pump 4b and PSD-95 could be co-immunoprecipitated from COS-7 cells overexpressing these proteins. Surface plasmon resonance revealed that a C-terminal pump 4b peptide interacted with the PDZ1 + 2 domains of hDlg with nanomolar affinity (KD = 1.6 nM), whereas binding to PDZ3 was in the micromolar range (KD = 1.2 microM). In contrast, the corresponding C-terminal peptide of Ca2+ pump 2b interacted weakly with PDZ1 + 2 and not at all with PDZ3 of hDlg. Ca2+ pump 4b bound strongly to PDZ1 + 2 + 3 of hDlg on filter assays, whereas isoform 2b bound weakly, and the splice variants 2a and 4a failed to bind. Together, these data demonstrate a direct physical binding of Ca2+ pump isoform 4b to MAGUKs via their PDZ domains and reveal a novel role of alternative splicing within the family of plasma membrane Ca2+ pumps. Alternative splicing may dictate their specific interaction with PDZ domain-containing proteins, potentially influencing their localization and incorporation into functional multiprotein complexes at the plasma membrane.

Seizure-induced alterations of plasma membrane calcium ATPase isoforms 1, 2 and 3 mRNA and protein in rat hippocampus.

Improper intracellular regulation of the ubiquitous second messenger, calcium, has been linked to several pathological conditions. The plasma membrane calcium ATPase (PMCA) is one of the primary systems for translocating calcium from the cytosol to the extracellular milieu. As an initial assessment of the possible involvement of PMCAs in kainate (KA)-induced neurodegeneration, we have determined the effect of KA-induced seizures upon PMCA mRNA and protein. In situ hybridization was performed on tissue from adult male Sprague-Dawley rats sacrificed at various time points following i.p. injection of KA. KA altered the expression within the hippocampal subfields for mRNAs of PMCA isoforms 1 and 2. PMCA 1 and 2 mRNAs exhibited hybridization below control levels 12-48 h post-injection within CA1 and CA3. Within the dentate gyrus, PMCA 2 mRNA hybridized below control levels 4 h post-injection, but recovered to control levels by 24 h post-injection. Alterations in combined PMCA protein levels occurred at all time points examined post-injection. These observations provide evidence that KA-induced seizures alter the PMCAs at the mRNA and protein levels, suggesting a possible role for this calcium efflux system in the neuronal degeneration inherent to this paradigm.

mRNA expression of the four isoforms of the human plasma membrane Ca(2+)-ATPase in the human hippocampus.

Ca2+ dyshomeostasis is a contributing factor to the development and progression of neurodegenerative disease. Plasma membrane Ca(2+)-ATPases (PMCAs) are responsible for setting intracellular Ca2+ levels and may be involved in the dynamic processing of Ca2+ loads in normal and pathological conditions. In situ hybridization was employed to determine the expression pattern of the four human PMCA isoforms in the human hippocampus. PMCA1 and 3 mRNAs were weakly expressed throughout the hippocampal formation, whereas PMCA2 and 4 mRNA expression showed distinct regional differences, with increased levels in CA2 and the dentate gyrus. Differential expression of PMCA isoforms may reflect cellular differences in Ca(2+)-handling properties and provide a partial explanation for the differential susceptibility of hippocampal neurons to Ca(2+)-mediated cell death.

Application of a chimeric green fluorescent protein to study protein-protein interactions.

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria is an emerging tool to monitor gene expression in situ and in vivo. Because of its fluorescence properties, when GFP is fused in-frame to a specific protein of interest, various aspects of the behavior of this protein can be analyzed noninvasively. Here we describe a fusion between GFP and human calmodulin-like protein (CLP) and show that this protein retains fluorescence and known characteristics of CLP, including Ca(2+)-dependent interaction with phenyl-Sepharose and interaction with a specific cellular target protein. The results suggest a novel application for GFP fusion proteins in the rapid, nonradioactive detection of interacting proteins on gel overlays.