Presenilin (PS) genes linked to early-onset familialAlzheimer's disease encode polytopic membrane proteins that are presumed to constitute the catalytic subunit of ␥-secretase, forming a high molecular weight complex with other proteins. During our attempts to identify binding partners of PS2, we cloned CALP (calsenilin-like protein)/KChIP4, a novel member of calsenilin/ KChIP protein family that interacts with the C-terminal region of PS. Upon co-expression in cultured cells, CALP was directly bound to and co-localized with PS2 in endoplasmic reticulum. Alzheimer's disease (AD)1 is a progressive dementing neurodegenerative disorder characterized by a massive deposition of ␤-amyloid and tau-rich neurofibrillary lesions in the brains (reviewed in Ref. 1 and references therein). A subset of AD is inherited as an autosomal dominant trait, and mutations in three different genes have thus far been linked to early-onset autosomal dominant forms of familial AD (FAD). Among these, presenilin 1 (PS1) and PS2 account for the majority of the early onset FAD (1). PS1 and PS2 genes encode polytopic integral membrane proteins that are predominantly localized in intracellular membranes and span the membrane six to eight times.PS proteins undergo endoproteolysis to give rise to N-and C-terminal fragments, which are the preponderant forms of endogenous PS in vivo (2). These fragments form a heterodimer and are incorporated into high molecular weight (HMW) protein complexes (2-5) that are highly stabilized (t1 ⁄2 ϭ ϳ20 h; Ref.6), whereas holoproteins of PS are rapidly degraded (t1 ⁄2 ϭ ϳ2 h) (6, 7). The steady-state levels of PS fragments seem to be tightly regulated by competition for shared, but limiting, cellular factors, because overexpression of PS in transfected cells does not increase the overall level of PS fragments and replaces endogenous PS (8).PS plays an important role in the generation of amyloid ␤ peptides (A␤) by facilitating intramembranous ␥-cleavage of ␤-amyloid protein precursor (␤APP), as evidenced by the lack of A␤ production and accumulation of ␤APP C-terminal stubs in cells established from PS-null mice (9 -11). In contrast, FADlinked mutations in PS increase the production of highly fibrillogenic A␤42 (12-15), which is the initial and predominantly deposited A␤ species in AD brains (16, 17) and normally consists of only ϳ10% of total secreted A␤ (18). Moreover, genetic studies in invertebrates and PS-null mice suggested that ␥-cleavage-like proteolytic cleavage at site 3 to release Notch intracellular domain (NICD), which is the prerequisite for Notch signaling (reviewed in Ref. 19), also is facilitated by PS. Furthermore, recent findings that the two intramembranous aspartates within the 6th and 7th transmembrane (TM) domains of PS are required for ␥-secretase activities (20) and that transition state analogue ␥-secretase inhibitors specifically label PS fragments (21-24) strongly support the notion that the PS-containing macroprotein complex catalyzes ␥-cleavage and that PS may represent the catalytic ...

Background: TRPA1 forms Ca 2+ - and Zn 2+ -permeable ion channels that sense noxious substances. Results: TNF-α and IL1-α induce TRPA1 gene expression via nuclear factor-κB signaling and downstream activation of HIF1α. Conclusion: HIF1α links inflammatory mediators to ion channel expression. Significance: HIF1α acts by binding to a specific hypoxia response element-like motif and its flanking regions in the TRPA1 gene.

In the heart, fibroblasts play an essential role in the deposition of the extracellular matrix and they also secrete a number of hormonal factors. Although natriuretic peptides, including C-type natriuretic peptide (CNP) and brain natriuretic peptide, have antifibrotic effects on cardiac fibroblasts, the effects of CNP on fibroblast electrophysiology have not been examined. In this study, acutely isolated ventricular fibroblasts from the adult rat were used to measure the effects of CNP (2 × 10 −8 M) under whole-cell voltage-clamp conditions. CNP, as well as the natriuretic peptide C receptor (NPR-C) agonist cANF (2 × 10 −8 M), significantly increased an outwardly rectifying non-selective cation current (NSCC). This current has a reversal potential near 0 mV. Activation of this NSCC by cANF was abolished by pre-treating fibroblasts with pertussis toxin, indicating the involvement of G i proteins. The cANF-activated NSCC was inhibited by the compounds Gd 3+ , SKF 96365 and 2-aminoethoxydiphenyl borate. Quantitative RT-PCR analysis of mRNA from rat ventricular fibroblasts revealed the expression of several transient receptor potential (TRP) channel transcripts. Additional electrophysiological analysis showed that U73122, a phospholipase C antagonist, inhibited the cANF-activated NSCC. Furthermore, the effects of CNP and cANF were mimicked by the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG), independently of protein kinase C activity. These are defining characteristics of specific TRPC channels. More detailed molecular analysis confirmed the expression of full-length TRPC2, TRPC3 and TRPC5 transcripts. These data indicate that CNP, acting via the NPR-C receptor, activates a NSCC that is at least partially carried by TRPC channels in cardiac fibroblasts.

In articular cartilage inflammation, histamine release from mast cells is a key event. It can enhance cytokine production and matrix synthesis and also promote cell proliferation by stimulating chondrocytes. In this study, the functional impact of Ca2+-activated K+ (KCa) channels in the regulation of intracellular Ca2+ concentration ([Ca2+]i) in chondrocytes in response to histamine was examined using OUMS-27 cells, as a model of chondrocytes derived from human chondrosarcoma. Application of histamine induced a significant [Ca2+]i rise and also membrane hyperpolarization, and both effects were mediated by the stimulation of H1 receptors. The histamine-induced membrane hyperpolarization was attenuated to ∼50% by large-conductance KCa (BK) channel blockers, and further reduced by intermediate (IK) and small conductance KCa (SK) channel blockers. The tonic component of histamine-induced [Ca2+]i rise strongly depended on the presence of extracellular Ca2+ ([Ca2+]o) and was markedly reduced by La3+ or Gd3+ but not by nifedipine. It was significantly attenuated by BK channel blockers, and further blocked by the cocktail of BK, IK, and SK channel blockers. The KCa blocker cocktail also significantly reduced the store-operated Ca2+ entry (SOCE), which was induced by Ca2+ addition after store-depletion by thapsigargin in [Ca2+]o free solution. Our results demonstrate that the histamine-induced membrane hyperpolarization in chondrocytes due to KCa channel activation contributes to sustained Ca2+ entry mainly through SOCE channels in OUMS-27 cells. Thus, KCa channels appear to play an important role in the positive feedback mechanism of [Ca2+]i regulation in chondrocytes in the presence of articular cartilage inflammation.

The sesquiterpene (−)Englerin A (EA) is an organic compound from the plant Phyllanthus engleri which acts via heteromeric TRPC4/C1 channels to cause cytotoxicity in some types of cancer cell but not normal cells. Here we identified selective cytotoxicity of EA in human synovial sarcoma cells (SW982 cells) and investigated the mechanism. EA induced cation channel current (Icat) in SW982 cells with biophysical characteristics of heteromeric TRPC4/C1 channels. Inhibitors of homomeric TRPC4 channels were weak inhibitors of the Icat and EA-induced cytotoxicity whereas a potent inhibitor of TRPC4/C1 channels (Pico145) strongly inhibited Icat and cytotoxicity. Depletion of TRPC1 converted Icat into a current with biophysical and pharmacological properties of homomeric TRPC4 channels and depletion of TRPC1 or TRPC4 suppressed the cytotoxicity of EA. A Na+/K+-ATPase inhibitor (ouabain) potentiated EA-induced cytotoxicity and direct Na+ loading by gramicidin-A caused Pico145-resistant cytotoxicity in the absence of EA. We conclude that EA has a potent cytotoxic effect on human synovial sarcoma cells which is mediated by heteromeric TRPC4/C1 channels and Na+ loading.