The cytosolic N-terminus of presenilin-1 potentiates mouse ryanodine receptor single channel activity

Ryanodine receptors (RyRs) amplify intracellular Ca(2+) signals by massively releasing Ca(2+) from intracellular stores. Exaggerated chronic Ca(2+) release can trigger cellular apoptosis underlying a variety of neurodegenerative diseases. Aberrant functioning of presenilin-1 (PS1) protein instigates Ca(2+)-dependent apoptosis, providing a basis for the "calcium hypothesis" of Alzheimer's disease (AD). To get insight into this problem, we hypothesized that the previously reported physical interaction between RyR and PS1 modulates functional properties of the RyR. We generated a soluble cytoplasmic N-terminal fragment of PS1 comprising the first 82 amino acid (PS1 NTF(1-82)), the candidate for interaction with putative cytoplasmic modulatory sites of the RyR, and studied its effect on single channel currents of mouse brain RyRs incorporated in lipid bilayers. PS1 NTF(1-82) strongly increased both mean currents (EC(50)=12nM, Hill coefficient (n(H)) approximately 1) and open probability for higher sublevels for single RyR channels (EC(50)=7nM, n(H) approximately 2). Bell-shaped Ca(2+)-activation curve remained unchanged, suggesting that PS1 NTF(1-82) allosterically potentiates RyRs, but that the channel still requires Ca(2+) for activation. Corroborating such an independent mechanism, the RyR potentiation by PS1 NTF(1-82) was overridden by receptor desensitization at high [Ca(2+)] (pCa>5). This potentiation of RyR by PS1 NTF(1-82) reveals a new mechanism of physiologically relevant PS1-regulated Ca(2+) release from intracellular stores, which could be alternative or additional to recently reported intracellular Ca(2+) leak channels formed by PS1 holoproteins.     

Contributions of Ca2+ to galectin-1-induced exposure of phosphatidylserine on activated neutrophils

Apoptotic cells redistribute phosphatidylserine (PS) to the cell surface by both Ca(2+)-dependent and -independent mechanisms. Binding of dimeric galectin-1 (dGal-1) to glycoconjugates on N-formyl-Met-Leu-Phe (fMLP)-activated neutrophils exposes PS and facilitates neutrophil phagocytosis by macrophages, yet it does not initiate apoptosis. We asked whether dGal-1 initiated Ca(2+) fluxes that are required to redistribute PS to the surface of activated neutrophils. Prolonged occupancy by dGal-1 was required to maximally mobilize PS to the surfaces of fMLP-activated neutrophils. Like fMLP, dGal-1 rapidly elevated cytosolic Ca(2+) levels in Fluo-4-loaded neutrophils. An initial Ca(2+) mobilization from intracellular stores was followed by movement of extracellular Ca(2+) to the cytosolic compartment, with return to basal Ca(2+) levels within 10 min. Chelation of extracellular Ca(2+) did not prevent PS mobilization. Chelation of intracellular Ca(2+) revealed that fMLP and dGal-1 independently release Ca(2+) from intracellular stores that cooperate to induce optimal redistribution of PS. Ca(2+) mobilization by ionomycin did not permit dGal-1 to mobilize PS, indicating that fMLP initiated both Ca(2+)-dependent and -independent signals that facilitated dGal-1-induced exposure of PS. dGal-1 elevated cytosolic Ca(2+) and mobilized PS through a pathway that required action of Src kinases and phospholipase Cgamma. These results demonstrate that transient Ca(2+) fluxes contribute to a sustained redistribution of PS on neutrophils activated with fMLP and dGal-1.     

The role of the presenilin-1 homologue gene sel-12 of Caenorhabditis elegans in apoptotic activities

Many cases of autosomal dominant early onset familial Alzheimer's disease result from mutations in presenilin-1 (PS1). In this study, we examined the role of the PS1 homologue gene sel-12 of Caenorhabditis elegans under oxidative stress and clarified the sel-12-induced apoptosis. A genetic null allele mutant, sel-12(ar171), showed resistance to oxidative stress and prevented mitochondrial dysfunction-induced apoptosis. On the other hand, another allele mutant, sel-12(ar131), that carries a missense mutation showed a proapoptotic activity, which may be the result of a gain of function property. Also, sel-12(ar131)-induced apoptosis was ced-3- and ced-4-dependent. Dantrolene, which specifically inhibits Ca(2+) release from endoplasmic reticulum stores, prevents sel-12(ar131)-induced apoptosis. SEL-12, which is localized in the endoplasmic reticulum, may induce apoptosis through abnormal calcium release from the endoplasmic reticulum. Together, with the previous finding that human PS1 could substitute for SEL-12, these results suggest the similar involvement of PS1-inducing apoptosis under oxidative stress and mitochondrial dysfunction in the Alzheimer's Disease brain.     

Glucose and endoplasmic reticulum calcium channels regulate HIF-1beta via presenilin in pancreatic beta-cells

Pancreatic beta-cell death is a critical event in type 1 diabetes, type 2 diabetes, and clinical islet transplantation. We have previously shown that prolonged block of ryanodine receptor (RyR)-gated release from intracellular Ca(2+) stores activates calpain-10-dependent apoptosis in beta-cells. In the present study, we further characterized intracellular Ca(2+) channel expression and function in human islets and the MIN6 beta-cell line. All three RyR isoforms were identified in human islets and MIN6 cells, and these endoplasmic reticulum channels were observed in close proximity to mitochondria. Blocking RyR channels, but not sarco/endoplasmic reticulum ATPase (SERCA) pumps, reduced the ATP/ADP ratio. Blocking Ca(2+) flux through RyR or inositol trisphosphate receptor channels, but not SERCA pumps, increased the expression of hypoxia-inducible factor (HIF-1beta). Moreover, inhibition of RyR or inositol trisphosphate receptor channels, but not SERCA pumps, increased the expression of presenilin-1. Both HIF-1beta and presenilin-1 expression were also induced by low glucose. Overexpression of presenilin-1 increased HIF-1beta, suggesting that HIF is downstream of presenilin. Our results provide the first evidence of a presenilin-HIF signaling network in beta-cells. We demonstrate that this pathway is controlled by Ca(2+) flux through intracellular channels, likely via changes in mitochondrial metabolism and ATP. These findings provide a mechanistic understanding of the signaling pathways activated when intracellular Ca(2+) homeostasis and metabolic activity are suppressed in diabetes and islet transplantation.     

Ca2+-independent binding and cellular expression profiles question a significant role of calmyrin in transduction of Ca2+-signals to Alzheimer's disease-related presenilin 2 in forebrain

The interaction between the EF-hand Ca(2+)-binding protein calmyrin and presenilin 2 (PS2) has been suggested to play a role in Alzheimer's disease (AD). We now report that calmyrin binds specifically endogenous PS2 and not PS1. However, binding appears to be Ca(2+)-independent and calmyrin does not exhibit a Ca(2+)-dependent translocation to intracellular membranes as demonstrated in a Ca(2+)-myristoyl switch assay. Moreover, calmyrin is only present at very low levels in brain areas associated with the onset of AD. In rat, forebrain calmyrin is localized only in a subset of principal neurons, similarly as in human forebrain. Finally, subcellular fractionation demonstrates only a limited overlap of calmyrin and PS2 at neuronal membranes. We therefore conclude that calmyrin will not contribute significantly as a Ca(2+)-sensor that transduces Ca(2+)-signaling events to PS2 in forebrain.     

Presenilin 2 interacts with sorcin, a modulator of the ryanodine receptor

Perturbed Ca(2+) homeostasis is a common molecular consequence of familial Alzheimer's disease-linked presenilin mutations. We report here the molecular interaction of the large hydrophilic loop region of presenilin 2 (PS2) with sorcin, a penta-EF-hand Ca(2+)-binding protein that serves as a modulator of the ryanodine receptor intracellular Ca(2+) channel. The association of endogenous sorcin and PS2 was demonstrated in cultured cells and human brain tissues. Membrane-associated sorcin and a subset of the functional PS2 complexes were co-localized to a novel subcellular fraction that is distinctively positive for calcineurin B. Sorcin was found to interact with PS2 endoproteolytic fragments but not full-length PS2, and the sorcin/PS2 interaction was greatly enhanced by treatment with the Ca(2+) ionophore A23187. Our findings reveal a molecular link between PS2 and intracellular Ca(2+) channels (i.e. ryanodine receptor) and substantiate normal and/or pathological roles of PS2 in intracellular Ca(2+) homeostasis.     

Lack of evidence for presenilins as endoplasmic reticulum Ca2+ leak channels

Familial Alzheimer disease (FAD) is linked to mutations in the presenilin (PS) homologs. FAD mutant PS expression has several cellular consequences, including exaggerated intracellular Ca(2+) ([Ca(2+)](i)) signaling due to enhanced agonist sensitivity and increased magnitude of [Ca(2+)](i) signals. The mechanisms underlying these phenomena remain controversial. It has been proposed that PSs are constitutively active, passive endoplasmic reticulum (ER) Ca(2+) leak channels and that FAD PS mutations disrupt this function resulting in ER store overfilling that increases the driving force for release upon ER Ca(2+) release channel opening. To investigate this hypothesis, we employed multiple Ca(2+) imaging protocols and indicators to directly measure ER Ca(2+) dynamics in several cell systems. However, we did not observe consistent evidence that PSs act as ER Ca(2+) leak channels. Nevertheless, we confirmed observations made using indirect measurements employed in previous reports that proposed this hypothesis. Specifically, cells lacking PS or expressing a FAD-linked PS mutation displayed increased area under the ionomycin-induced [Ca(2+)](i) versus time curve (AI) compared with cells expressing WT PS. However, an ER-targeted Ca(2+) indicator revealed that this did not reflect overloaded ER stores. Monensin pretreatment selectively attenuated the AI in cells lacking PS or expressing a FAD PS allele. These findings contradict the hypothesis that PSs form ER Ca(2+) leak channels and highlight the need to use ER-targeted Ca(2+) indicators when studying ER Ca(2+) dynamics.     

Effects of chronic hypoxia on Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells.

Microfluorimetric measurements of intracellular calcium ion concentration [Ca(2+)](i) were employed to examine the effects of chronic hypoxia (2.5% O(2), 24 h) on Ca(2+) stores and capacitative Ca(2+) entry in human neuroblastoma (SH-SY5Y) cells. Activation of muscarinic receptors evoked rises in [Ca(2+)](i) which were enhanced in chronically hypoxic cells. Transient rises of [Ca(2+)](i) evoked in Ca(2+)-free solutions were greater and decayed more slowly following exposure to chronic hypoxia. In control cells, these transient rises of [Ca(2+)](i) were also enhanced and slowed by removal of external Na(+), whereas the same manoeuvre did not affect responses in chronically hypoxic cells. Capacitative Ca(2+) entry, observed when re-applying Ca(2+) following depletion of intracellular stores, was suppressed in chronically hypoxic cells. Western blots revealed that presenilin-1 levels were unaffected by chronic hypoxia. Exposure of cells to amyloid beta peptide (1-40) also increased transient [Ca(2+)](i) rises, but did not mimic any other effects of chronic hypoxia. Our results indicate that chronic hypoxia causes increased filling of intracellular Ca(2+) stores, suppressed expression or activity of Na(+)/Ca(2+) exchange and reduced capacitative Ca(2+) entry. These effects are not attributable to increased amyloid beta peptide or presenilin-1 levels, but are likely to be important in adaptive cellular remodelling in response to prolonged hypoxic or ischemic episodes.     

Calcium binding sequences in calmyrin regulates interaction with presenilin-2

Calmyrin is a myristoylated calcium binding protein that contains four putative EF-hands. Calmyrin interacts with a number of proteins, including presenilin-2 (PS2). However, the biophysical properties of calmyrin, and the molecular mechanisms that regulate its binding to different partners, are not well understood. By site-directed mutagenesis and Ca2+ binding studies, we found that calmyrin binds two Ca2+ ions with a dissociation constant of approximately 53 microM, and that the two C-terminal EF-hands 3 and 4 bind calcium. Using ultraviolet spectroscopy, circular dichroism (CD), and NMR, we found that Ca(2+)-free and -bound calmyrin have substantially different protein conformations. By yeast two-hybrid assays, we found that both EF-hands 3 and 4 of calmyrin must be intact for calmyrin to interact with PS2-loop sequences. Pulse-chase studies of HeLa cells transfected with calmyrin expression constructs indicated that wild-type (Wt) calmyrin has a half-life of approximately 75 min, whereas a mutant defective in myristoylation turns over more rapidly (half-life of 35 min). By contrast, the half-lives of calmyrin mutants with a disrupted EF-hand 3 or EF-hand 4 were 52 and 170 min, respectively. Using immunofluorescence staining of HeLa cells transfected with Wt and mutant calmyrin cDNAs, we found that both calcium binding and myristoylation are important for dynamic intracellular targeting of calmyrin. Double immunofluorescence microscopy indicated that Wt and myristoylation-defective calmyrin proteins colocalize efficiently and to the same extent with PS2, whereas calmyrin mutants defective in calcium binding display less colocalization with PS2. Our results suggest that calmyrin functions as a calcium sensor and that calcium binding sequences in calmyrin are important for interaction with the PS2 loop.     

The N-terminus of presenilin-2 increases single channel activity of brain ryanodine receptors through direct protein-protein interaction

Presenilin-1 (PS1) and presenilin-2 (PS2) form the catalytic core in gamma-secretase complexes and mutations in these proteins result in aberrant cleavage of amyloid precursor protein leading to accumulation of the beta-amyloid in the brain of familial Alzheimer Disease patients. PS2 possesses a hydrophilic cytoplasmic N-terminal domain (PS2 NTF1-87) dispensable for gamma-secretase activity with physiological functions yet to be determined. The effects of this soluble 87 amino acid fragment of mouse PS2 on single channel activity of mouse brain ryanodine receptors (RyR) were determined. PS2 NTF1-87 application to the cytoplasmic side of the RyR significantly increased single channel activity by favoring higher sublevel openings. The Ca(2+) activation and desensitization ranges for RyRs were unchanged. We demonstrate facilitation of RyR gating by PS2 NTF1-87, which might represent a general mechanism of RyR regulation by presenilins potentially prone to be affected by mutations or external stimuli contributing to the development of neurodegenerative diseases.     

Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer's disease-linked mutations

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder. Mutations in presenilins 1 and 2 (PS1 and PS2) account for approximately 40% of familial AD (FAD) cases. FAD mutations and genetic deletions of presenilins have been associated with calcium (Ca(2+)) signaling abnormalities. We demonstrate that wild-type presenilins, but not PS1-M146V and PS2-N141I FAD mutants, can form low-conductance divalent-cation-permeable ion channels in planar lipid bilayers. In experiments with PS1/2 double knockout (DKO) mouse embryonic fibroblasts (MEFs), we find that presenilins account for approximately 80% of passive Ca(2+) leak from the endoplasmic reticulum. Deficient Ca(2+) signaling in DKO MEFs can be rescued by expression of wild-type PS1 or PS2 but not by expression of PS1-M146V or PS2-N141I mutants. The ER Ca(2+) leak function of presenilins is independent of their gamma-secretase activity. Our data suggest a Ca(2+) signaling function for presenilins and provide support for the "Ca(2+) hypothesis of AD."     

Presenilin-1 forms complexes with the cadherin/catenin cell-cell adhesion system and is recruited to intercellular and synaptic contacts

In MDCK cells, presenilin-1 (PS1) accumulates at intercellular contacts where it colocalizes with components of the cadherin-based adherens junctions. PS1 fragments form complexes with E-cadherin, beta-catenin, and alpha-catenin, all components of adherens junctions. In confluent MDCK cells, PS1 forms complexes with cell surface E-cadherin; disruption of Ca(2+)-dependent cell-cell contacts reduces surface PS1 and the levels of PS1-E-cadherin complexes. PS1 overexpression in human kidney cells enhances cell-cell adhesion. Together, these data show that PS1 incorporates into the cadherin/catenin adhesion system and regulates cell-cell adhesion. PS1 concentrates at intercellular contacts in epithelial tissue; in brain, it forms complexes with both E- and N-cadherin and concentrates at synaptic adhesions. That PS1 is a constituent of the cadherin/catenin complex makes that complex a potential target for PS1 FAD mutations.     

Interaction of Alzheimer's presenilin-1 and presenilin-2 with Bcl-X(L). A potential role in modulating the threshold of cell death.

The familial Alzheimer's disease gene products, presenilin-1 and presenilin-2, have been reported to be functionally involved in amyloid precursor protein processing, notch receptor signaling, and programmed cell death or apoptosis. However, the molecular mechanisms by which presenilins regulate these processes remain unknown. With regard to the latter, we describe a molecular link between presenilins and the apoptotic pathway. Bcl-X(L), an anti-apoptotic member of the Bcl-2 family was shown to interact with the carboxyl-terminal fragments of PS1 and PS2 by the yeast two-hybrid system. In vivo interaction analysis revealed that both PS2 and its naturally occurring carboxyl-terminal products, PS2short and PS2Ccas, associated with Bcl-X(L), whereas the caspase-3-generated amino-terminal PS2Ncas fragment did not. This interaction was corroborated by demonstrating that Bcl-X(L) and PS2 partially co-localized to sites of the vesicular transport system. Functional analysis revealed that presenilins can influence mitochondrial-dependent apoptotic activities, such as cytochrome c release and Bax-mediated apoptosis. Together, these data support a possible role of the Alzheimer's presenilins in modulating the anti-apoptotic effects of Bcl-X(L).     

Capacitive calcium entry is directly attenuated by mutant presenilin-1, independent of the expression of the amyloid precursor protein

Mutant presenilin-1 (PS1) increases amyloid peptide production, attenuates capacitative calcium entry (CCE), and augments calcium release from the endoplasmatic reticulum (ER). Here we measured the intracellular free Ca(2+) concentration in hippocampal neurons from six different combinations of transgenic and gene-ablated mice to demonstrate that mutant PS1 attenuated CCE directly, independent of the expression of the amyloid precursor protein (APP). On the other hand, increased Ca(2+) release from the ER in mutant PS1 neurons, as induced by thapsigargin, was clearly dependent on the presence of APP and its processing by PS1, i.e. on the generation of the amyloid peptides and the APP C99 fragments. This observation was corroborated by the thapsigargin-induced increase in cytosolic [Ca(2+)](i) in PS1 deficient neurons, which accumulate C99 fragments due to deficient gamma-secretase activity. Moreover, co-expression of mutant APP[V717I] in PS1-deficient neurons further increased the apparent size of the ER calcium stores in parallel with increasing levels of the APP processing products. We conclude that mutant PS1 deregulates neuronal calcium homeostasis by two different actions: (i) direct attenuation of CCE at the cell-surface independent of APP; and (ii) indirect increase of ER-calcium stores via processing of APP and generation of amyloid peptides and C99 fragments.     

Involvement of sodium in early phosphatidylserine exposure and phospholipid scrambling induced by P2X7 purinoceptor activation in thymocytes

Extracellular ATP (ATP(ec)), a possible effector in thymocyte selection, induces thymocyte death via purinoceptor activation. We show that ATP(ec) induced cell death by apoptosis, rather than lysis, and early phosphatidylserine (PS) exposure and phospholipid scrambling in a limited thymocyte population (35-40%). PS externalization resulted from the activation of the cationic channel P2X7 (formerly P2Z) receptor and was triggered in all thymocyte subsets although to different proportions in each one. Phospholipid movement was dependent on ATP(ec)-induced Ca(2+) and/or Na(+) influx. At physiological external Na(+) concentration, without external Ca(2+), PS was exposed in all ATP(ec)-responsive cells. In contrast, without external Na(+), physiological external Ca(2+) concentration promoted a submaximal response. Altogether these data show that Na(+) influx plays a major role in the rapid PS exposure induced by P2X7 receptor activation in thymocytes.     

Activation of phospholipase C delta1 through C2 domain by a Ca(2+)-enzyme-phosphatidylserine ternary complex.

The concentration of free Ca(2+) and the composition of nonsubstrate phospholipids profoundly affect the activity of phospholipase C delta1 (PLCdelta1). The rate of PLCdelta1 hydrolysis of phosphatidylinositol 4,5-bisphosphate was stimulated 20-fold by phosphatidylserine (PS), 4-fold by phosphatidic acid (PA), and not at all by phosphatidylethanolamine or phosphatidylcholine (PC). PS reduced the Ca(2+) concentration required for half-maximal activation of PLCdelta1 from 5.4 to 0.5 microM. In the presence of Ca(2+), PLCdelta1 specifically bound to PS/PC but not to PA/PC vesicles in a dose-dependent and saturable manner. Ca(2+) also bound to PLCdelta1 and required the presence of PS/PC vesicles but not PA/PC vesicles. The free Ca(2+) concentration required for half-maximal Ca(2+) binding was estimated to be 8 microM. Surface dilution kinetic analysis revealed that the K(m) was reduced 20-fold by the presence of 25 mol % PS, whereas V(max) and K(d) were unaffected. Deletion of amino acid residues 646-654 from the C2 domain of PLCdelta1 impaired Ca(2+) binding and reduced its stimulation and binding by PS. Taken together, the results suggest that the formation of an enzyme-Ca(2+)-PS ternary complex through the C2 domain increases the affinity for substrate and consequently leads to enzyme activation.     

Membrane targeting of C2 domains of phospholipase C-delta isoforms.

The C2 domain is a Ca(2+)-dependent membrane-targeting module found in many cellular proteins involved in signal transduction or membrane trafficking. To understand the mechanisms by which the C2 domain mediates the membrane targeting of PLC-delta isoforms, we measured the in vitro membrane binding of the C2 domains of PLC-delta1, -delta3, and -delta4 by surface plasmon resonance and monolayer techniques and their subcellular localization by time-lapse confocal microscopy. The membrane binding of the PLC-delta1-C2 is driven by nonspecific electrostatic interactions between the Ca(2+)-induced cationic surface of protein and the anionic membrane and specific interactions involving Ca(2+), Asn(647), and phosphatidylserine (PS). The PS selectivity of PLC-delta1-C2 governs its specific Ca(2+)-dependent subcellular targeting to the plasma membrane. The membrane binding of the PLC-delta3-C2 also involves Ca(2+)-induced nonspecific electrostatic interactions and PS coordination, and the latter leads to specific subcellular targeting to the plasma membrane. In contrast to PLC-delta1-C2 and PLC-delta3-C2, PLC-delta4-C2 has significant Ca(2+)-independent membrane affinity and no PS selectivity due to the presence of cationic residues in the Ca(2+)-binding loops and the substitution of Ser for the Ca(2+)-coordinating Asp in position 717. Consequently, PLC-delta4-C2 exhibits unique pre-localization to the plasma membrane prior to Ca(2+) import and non-selective Ca(2+)-mediated targeting to various cellular membranes, suggesting that PLC-delta4 might have a novel regulatory mechanism. Together, these results establish the C2 domains of PLC-delta isoforms as Ca(2+)-dependent membrane targeting domains that have distinct membrane binding properties that control their subcellular localization behaviors.     

Effects of Ca2+ and EGTA on P680*+ reduction kinetics and O2 evolution of Photosystem II

We report for the first time significant changes in the P680*+ reduction kinetics of Photosystem II (PS II) in which the 17 and 23 kDa extrinsic polypeptides are intact, in the presence of Ca(2+) or ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) which were added to vary the Ca(2+) concentration from 5 microM to 30 mM. The decrease in the extent of normal P680*+ reduction decay with lifetimes of 40-370 ns and a corresponding increase in the extent of kinetics with lifetimes of 20-220 micros was interpreted as being due to electron transfer from Y(Z) to P680*+ being replaced by slow forward conduction and by processes including P680*+/Q(A)(-) recombination. The question of whether changes in P680*+ reduction kinetics were caused by loss of Ca(2+) from PS II or by direct interaction of EGTA with PS II was addressed by lowering the free-Ca(2+) concentration of suspensions of PS II core complexes by serial dilution in the absence of EGTA. Despite a significant decrease in the rate of O(2) evolution after this treatment, only small changes in the P680*+ reduction kinetics were observed. Loss of Ca(2+) did not affect P680*+ reduction associated with electron transfer from Y(Z). Since much larger changes in the P680*+ reduction kinetics of intact PS II occurred at comparable free-Ca(2+) concentrations in the presence of EGTA, we conclude that EGTA influenced the P680*+ reduction kinetics by directly interacting with PS II rather than by lowering the free Ca(2+) concentration of the surrounding media. Notwithstanding these effects, we show that useful information about Ca(2+) binding to PS II can be obtained when direct interaction of EGTA is taken into account.     

Receptor-operated Ca2+ entry mediated by TRPC3/TRPC6 proteins in rat prostate smooth muscle (PS1) cell line

Prostate smooth muscle cells predominantly express alpha1-adrenoceptors (alpha1-AR). alpha1-AR antagonists induce prostate smooth muscle relaxation and therefore they are useful therapeutic compounds for the treatment of benign prostatic hyperplasia symptoms. However, the Ca(2+) entry pathways associated with the activation of alpha1-AR in the prostate have yet to be elucidated. In many cell types, mammalian homologues of transient receptor potential (TRP) genes, first identified in Drosophila, encode TRPC (canonical TRP) proteins. They function as receptor-operated channels (ROCs) which are involved in various physiological processes such as contraction, proliferation, apoptosis, and differentiation. To date, the expression and function of TRPC channels have not been studied in prostate smooth muscle. In fura-2 loaded PS1 (a prostate smooth muscle cell line) which express endogenous alpha1A-ARs, alpha-agonists epinephrine (EPI), and phenylephrine (PHE) induced Ca(2+) influx which depended on the extracellular Ca(2+) and PLC activation but was independent of PKC activation. Thus, we have tested two membrane-permeable analogues of diacylglycerol (DAG), oleoyl-acyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG). They initiated Ca(2+) influx whose properties were similar to those induced by the alpha-agonists. Sensitivity to 2-aminoethyl diphenylborate (2-APB), SKF-96365 and flufenamate implies that Ca(2+)-permeable channels mediated both alpha-agonist- and OAG-evoked Ca(2+) influx. Following the sarcoplasmic reticulum (SR) Ca(2+) store depletion by thapsigargin (Tg), a SERCA inhibitor, OAG and PHE were both still able to activate Ca(2+) influx. However, OAG failed to enhance Ca(2+) influx when added in the presence of an alpha-agonist. RT-PCR and Western blotting performed on PS1 cells revealed the presence of mRNAs and the corresponding TRPC3 and TRPC6 proteins. Experiments using an antisense strategy showed that both alpha-agonist- and OAG-induced Ca(2+) influx required TRPC3 and TRPC6, whereas the Tg-activated ("capacitative") Ca(2+) entry involved only TRPC3 encoded protein. It may be thus concluded that PS1 cells express TRPC3 and TRPC6 proteins which function as receptor- and store-operated Ca(2+) entry pathways.