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PubMed2004

The canonical transient receptor potential 6 channel as a putative phosphatidylinositol 3,4,5-trisphosphate-sensitive calcium entry system.

We previously reported that phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), a lipid product of phosphoinositide 3-kinase (PI3K), induced Ca(2+) influx via a noncapacitative pathway in platelets, Jurkat T cells, and RBL-2H3 mast cells. The identity of this Ca(2+) influx system, however, remains unclear. Here, we investigate a potential link between PIP(3)-sensitive Ca(2+) entry and the canonical transient receptor potential (TRPC) channels by developing stable human embryonic kidney (HEK) 293 cell lines expressing TRPC1, TRPC3, TRPC5, and TRPC6. Two lines of evidence support TRPC6 as a putative target by which PIP(3) induces Ca(2+) influx. First, Fura-2 fluorometric Ca(2+) analysis shows the ability of PIP(3) to selectively stimulate [Ca(2+)](i) increase in TRPC6-expressing cells. Second, pull-down analysis indicates specific interactions between biotin-PIP(3) and TRPC6 protein. Our data indicate that PIP(3) activates store-independent Ca(2+) entry in TRPC6 cells via a nonselective cation channel. Although the activating effect of PIP(3) on TRPC6 is reminiscent to that of 1-oleoyl-2-acetyl-sn-glycerol, this activation is not attributable to the diacylglycerol substructure of PIP(3) since other phosphoinositides failed to trigger Ca(2+) responses. The PIP(3)-activated Ca(2+) entry is inhibited by known TRPC6 inhibitors such as Gd(3+) and SKF96365 and is independent of IP(3) production. Furthermore, we demonstrated that TRPC6 overexpression or antisense downregulation significantly alters the amplitude of PIP(3)- and anti-CD3-activated Ca(2+) responses in Jurkat T cells. Consequently, the link between TRPC6 and PIP(3)-mediated Ca(2+) entry provides a framework to account for an intimate relationship between PI3K and PLCgamma in initiating Ca(2+) response to agonist stimulation in T lymphocytes.

Biological Transport↗
PubMed2006

STIM1 carboxyl-terminus activates native SOC, I(crac) and TRPC1 channels.

Receptor-evoked Ca2+ signalling involves Ca2+ release from the endoplasmic reticulum, followed by Ca2+ influx across the plasma membrane. Ca2+ influx is essential for many cellular functions, from secretion to transcription, and is mediated by Ca2+-release activated Ca2+ (I(crac)) channels and store-operated calcium entry (SOC) channels. Although the molecular identity and regulation of I(crac) and SOC channels have not been precisely determined, notable recent findings are the identification of STIM1, which has been indicated to regulate SOC and I(crac) channels by functioning as an endoplasmic reticulum Ca2+ sensor, and ORAI1 (ref. 7) or CRACM1 (ref. 8)--both of which may function as I(crac) channels or as an I(crac) subunit. How STIM1 activates the Ca2+ influx channels and whether STIM1 contributes to the channel pore remains unknown. Here, we identify the structural features that are essential for STIM1-dependent activation of SOC and I(crac) channels, and demonstrate that they are identical to those involved in the binding and activation of TRPC1. Notably, the cytosolic carboxyl terminus of STIM1 is sufficient to activate SOC, I(crac) and TRPC1 channels even when native STIM1 is depleted by small interfering RNA. Activity of STIM1 requires an ERM domain, which mediates the selective binding of STIM1 to TRPC1, 2 and 4, but not to TRPC3, 6 or 7, and a cationic lysine-rich region, which is essential for gating of TRPC1. Deletion of either region in the constitutively active STIM1(D76A) yields dominant-negative mutants that block native SOC channels, expressed TRPC1 in HEK293 cells and I(crac) in Jurkat cells. These observations implicate STIM1 as a key regulator of activity rather than a channel component, and reveal similar regulation of SOC, I(crac) and TRPC channel activation by STIM1.

Amino Acid Sequence↗
PubMed2001

The transient receptor potential protein homologue TRP6 is the essential component of vascular alpha(1)-adrenoceptor-activated Ca(2+)-permeable cation channel.

The Drosophila transient receptor potential protein (TRP) and its mammalian homologues are thought to be Ca(2+)-permeable cation channels activated by G protein (G(q/11))-coupled receptors and are regarded as an interesting molecular model for the Ca(2+) entry mechanisms associated with stimulated phosphoinositide turnover and store depletion. However, there is little unequivocal evidence linking mammalian TRPs with particular native functions. In this study, we have found that heterologous expression of murine TRP6 in HEK293 cells reproduces almost exactly the essential biophysical and pharmacological properties of alpha(1)-adrenoceptor-activated nonselective cation channels (alpha(1)-AR-NSCC) previously identified in rabbit portal vein smooth muscle. Such properties include activation by diacylglycerol; S-shaped current-voltage relationship; high divalent cation permeability; unitary conductance of 25 to 30 pS and augmentation by flufenamate and Ca(2+); and blockade by Cd(2+), La(3+), Gd(3+), SK&F96365, and amiloride. Reverse transcriptase-polymerase chain reaction and confocal laser scanning microscopy using TRP6-specific primers and antisera revealed that the level of TRP6 mRNA expression was remarkably high in both murine and rabbit portal vein smooth muscles as compared with other TRP subtypes, and the immunoreactivity to TRP6 protein was localized near the sarcolemmal region of single rabbit portal vein myocytes. Furthermore, treatment of primary cultured portal vein myocytes with TRP6 antisense oligonucleotides resulted in marked inhibition of TRP6 protein immunoreactivity as well as selective suppression of alpha(1)-adrenoceptor-activated, store depletion-independent cation current and Ba(2+) influx. These results strongly indicate that TRP6 is the essential component of the alpha(1)-AR-NSCC, which may serve as a store depletion-independent Ca(2+) entry pathway during increased sympathetic activity.

Adrenergic alpha-Agonists↗
PubMedDate not supplied

Lysophosphatidylcholine, a component of atherogenic lipoproteins, induces the change of calcium mobilization via TRPC ion channels in cultured human corporal smooth muscle cells.

Hypercholesterolemia is a major risk factor for erectile dysfunction. To understand the mechanism(s) of hypercholesterolemia-induced erectile dysfunction, we studied the effect of lysophosphatidylcholine (LPC) on the membrane conductance of corporal smooth muscle cells. We used cultured human corporal smooth muscle cells. The intracelluar Ca2+ concentration ([Ca2+]i) and the influx of divalent cation was monitored by the ratio of fura-2 fluorescence (F(340/380)) and by the Mn2+-induced quenching rate of fura-2, respectively. The LPC-induced membrane current was characterized by the whole-cell patch-clamp technique and the molecular identity of suspected channels was probed by RT-PCR. LPC (20 microM) induced a statistically significant increase in F(340/380) to 119.9+/-3.9% of initial control (n=6) in corporal smooth muscle cells. The addition of 20 microM LPC accelerated the quenching rate of F360 by 59.5+/-11.8% (n=5). LPC activated nonselective cationic current (ILPC), similar to the known effects of phenylephrine in corporal myocytes. The size of ILPC at -60 mV was -55.3+/-6.3 pA (n=8). The transcript of transient receptor potential channel 6 (TRPC6) was detected in human corporal myocytes. We also found one splicing variant of TRPC6, TRPC6alpha. In conclusion, the present study suggests that the LPC, a major component of oxidized low-density lipoprotiens, increases calcium in corporal smooth muscle cells probably through activation of a TRPC6 channel and the increased [Ca2+]i by LPC via TRP channels is one of mechanisms for hypercholesterolemia-induced erectile dysfunction.

Alternative Splicing↗
PubMed2006

Permeation and selectivity of TRP channels.

Ion channels are pore-forming transmembrane proteins that allow ions to permeate biological membranes. Pore structure plays a crucial role in determining the ion permeation and selectivity properties of particular channels. In the past few decades, efforts have been undertaken to identify key elements of the pore regions of different classes of ion channels. In this review, we summarize current knowledge about permeation and selectivity of channel proteins from the transient receptor potential (TRP) superfamily. Whereas all TRP channels are permeable for cations, only two TRP channels are impermeable for Ca2+ (TRPM4, TRPM5), and two others are highly Ca2+ permeable (TRPV5, TRPV6). Despite the great advances in the TRP channel field during the past decade, only a limited number of reports have dealt with functional characterization of pore properties, biophysical aspects of cation permeation, or description of pore structures of TRP channels. This review gives an overview of available experimental and theoretical data and discusses the functional impact of pore-structure modifications on TRP channel properties.

Algorithms↗
PubMed2003

Receptor-coupled, DAG-gated Ca2+-permeable cationic channels in LNCaP human prostate cancer epithelial cells.

Although the prostate gland is a rich source of alpha1-adreno- (alpha1-AR) and m1-cholino receptors (m1-AChR), the membrane processes associated with their activation in glandular epithelial cells is poorly understood. We used the whole-cell patch-clamp technique to show that the agonists of the respective receptors, phenylephrine (PHE) and carbachol (CCh), activate cationic membrane currents in lymph node carcinoma of the prostate (LNCaP) human prostate cancer epithelial cells, which are not dependent on the filling status of intracellular IP3-sensitive Ca2+ stores, but directly gated by diacylglycerol (DAG), as evidenced by the ability of its membrane permeable analogue, OAG, to mimic the effects of the agonists. The underlying cationic channels are characterized by the weak field-strength Eisenman IV permeability sequence for monovalent cations (PK(25) > PCs(4.6) > PLi(1.4) > PNa(1.0)), and the following permeability sequence for divalent cations: PCa(1.0) > PMg(0.74) > PBa(0.6) > PSr(0.36) > PMn(0.3). They are 4.3 times more permeable to Ca2+ than Na+ and more sensitive to the inhibitor 2-APB than SK&F 96365. RT-PCR analysis shows that DAG-gated members of the transient receptor potential (TRP) channel family, including TRPC1 and TRPC3, are present in LNCaP cells. We conclude that, in prostate cancer epithelial cells, alpha1-ARs and m1-AChRs are functionally coupled to Ca2+-permeable DAG-gated cationic channels, for which TRPC1 and TRPC3 are the most likely candidates.

Base Sequence↗
PubMed2006

Mechanosensitive ion channels in skeletal muscle: a link in the membrane pathology of muscular dystrophy.

1. Mechanosensitive (MS) channels are expressed abundantly in skeletal muscle at all stages of development. In recordings from membrane patches, MS channels are constitutively active at the resting potential. The channels are selective for cations and have a large single-channel conductance (approximately 25 pS in physiological saline) and a high Ca2+ permeability (relative permeability of Ca2+ to K+ (PCa/PK) = 7). 2. Mechanosensitive channel activity recorded from the surface of myotubes from dystrophic mdx mice was substantially greater than the activity recorded from wild-type myotubes. Increased channel activity in the mutant results from the induction in a subpopulation of channels of a novel MS gating mode characterized by markedly prolonged channel openings and inactivation in response to membrane stretch. 3. Membrane stretch or a strong depolarization causes an irreversible switch to the stretch-inactivated gating mode in mdx myotubes. A stretch-induced shift in MS channel gating mode may contribute to stretch-induced elevations in [Ca2+]i during the early stages of disease pathogenesis. 4. Abnormalities of MS channel behaviour are also detected in recordings from patches on flexor digitorum brevis fibres acutely isolated from mdx mice. Mechanosensitive channel opening probability is higher in mdx fibres at all developmental stages. In addition, channel numbers are persistently elevated during postnatal development, failing to undergo a normal process of downregulation during the first 3 postnatal weeks. 5. Two distinct mechanisms may contribute to elevations of [Ca2+]i in dystrophin-deficient skeletal muscle: (i) a membrane stress-dependent switch of MS channels into to a prolonged opening mode; and (ii) a loss of developmental downregulation leading to persistent MS channel expression during postnatal muscle development.

Animals↗
PubMed2001

ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology.

Free ADP-ribose (ADPR), a product of NAD hydrolysis and a breakdown product of the calcium-release second messenger cyclic ADPR (cADPR), has no defined role as an intracellular signalling molecule in vertebrate systems. Here we show that a 350-amino-acid protein (designated NUDT9) and a homologous domain (NUDT9 homology domain) near the carboxy terminus of the LTRPC2/TrpC7 putative cation channel both function as specific ADPR pyrophosphatases. Whole-cell and single-channel analysis of HEK-293 cells expressing LTRPC2 show that LTRPC2 functions as a calcium-permeable cation channel that is specifically gated by free ADPR. The expression of native LTRPC2 transcripts is detectable in many tissues including the U937 monocyte cell line, in which ADPR induces large cation currents (designated IADPR) that closely match those mediated by recombinant LTRPC2. These results indicate that intracellular ADPR regulates calcium entry into cells that express LTRPC2.

Adenosine Diphosphate Ribose↗
PubMed2004

Complete RNAi rescue of neuronal degeneration in a constitutively active Drosophila TRP channel mutant.

RNA interference has been widely used to reduce the quantity of the proteins encoded by the targeted genes. A constitutively active, dominant allele of trp, TrpP365, causes massive degeneration of photoreceptors through a persistent and excessive Ca2+ influx. Here we show that a substantial reduction of the TRP channel protein by RNAi in TrpP365 heterozygotes completely rescues the neuronal degeneration and significantly improves the light-elicited responses of the eye. The reduction need not be complete, suggesting that rescue of degeneration may be possible with minimal side effects arising from overdepletion of the target protein.

Animals↗
PubMed2005

Pleckstrin homology domains: two halves make a hole?

In a recent issue of Nature, van Rossum et al. report binding of a "split" pleckstrin homology (PH) domain from phospholipase C-gamma(1) to the TRPC3 ion channel. Through sequence analyses and in vitro studies, they suggest a novel mode of protein-protein interaction in which two PH domain fragments in distinct proteins associate to form an "intermolecular" PH domain that binds inositol phospholipids and is required for ion channel location and function.

Animals↗
PubMed2006

TRPC3 and TRPC6 are essential for angiotensin II-induced cardiac hypertrophy.

Angiotensin (Ang) II participates in the pathogenesis of heart failure through induction of cardiac hypertrophy. Ang II-induced hypertrophic growth of cardiomyocytes is mediated by nuclear factor of activated T cells (NFAT), a Ca(2+)-responsive transcriptional factor. It is believed that phospholipase C (PLC)-mediated production of inositol-1,4,5-trisphosphate (IP(3)) is responsible for Ca(2+) increase that is necessary for NFAT activation. However, we demonstrate that PLC-mediated production of diacylglycerol (DAG) but not IP(3) is essential for Ang II-induced NFAT activation in rat cardiac myocytes. NFAT activation and hypertrophic responses by Ang II stimulation required the enhanced frequency of Ca(2+) oscillation triggered by membrane depolarization through activation of DAG-sensitive TRPC channels, which leads to activation of L-type Ca(2+) channel. Patch clamp recordings from single myocytes revealed that Ang II activated DAG-sensitive TRPC-like currents. Among DAG-activating TRPC channels (TRPC3, TRPC6, and TRPC7), the activities of TRPC3 and TRPC6 channels correlated with Ang II-induced NFAT activation and hypertrophic responses. These data suggest that DAG-induced Ca(2+) signaling pathway through TRPC3 and TRPC6 is essential for Ang II-induced NFAT activation and cardiac hypertrophy.

Angiotensin II↗
PubMed2007

Molecular determinants of TRP channel assembly.

Calcium channels play important roles in cellular signalling. TRP (transient receptor potential) channels form a superfamily of calcium channels through which Ca(2+) enters the cell. TRPs have six transmembrane segments with a putative pore between the fifth and the sixth segments, and assemble in tetrameric complexes to form functional Ca(2+) channels. They are thus similar to K(V) (voltage-gated potassium channel) channels in terms of structure and molecular determinants that promote subunit assembly. In this review, the molecular determinants mediating the assembly of Drosophila TRP, TRPC (TRP canonical), TRPV (TRP vanilloid) and K(V) channels are described.

Animals↗
PubMed2005

Molecular cloning of TRPC3a, an N-terminally extended, store-operated variant of the human C3 transient receptor potential channel.

AK032317 is the GenBank accession no. of a full-length RIKEN mouse cDNA. It encodes a putative variant of the C3-type TRPC (transient receptor potential channel) that differs from the previously cloned murine TRPC3 cDNA in that it has a 5' extension stemming from inclusion of an additional exon (exon 0). The extended cDNA adds 62 aa to the sequence of the murine TRPC3. Here, we report the cloning of a cDNA encoding the human homologue of this extended TRPC3 having a highly homologous 73-aa N-terminal extension, referred to as hTRPC3a. A query of the GenBank genomic database predicts the existence of a similar gene product also in rats. Transient expression of the longer TRPC3a in human embryonic kidney (HEK) cells showed that it mediates Ca2+ entry in response to stimulation of the Gq-phospholipase C beta pathway, which is similar to that mediated by the shorter hTRPC3. However, after isolation of HEK cells expressing hTRPC3 in stable form, TRPC3a gave rise to Ca2+-entry channels that are not only activated by the Gq-phospholipase C beta pathway (receptor-activated Ca entry) but also by thapsigargin triggered store depletion. In conjunction with findings from our and other laboratories that TRPC1, TRPC2, TRPC4, TRPC5, and TRPC7, can each mediate store-depletion-activated Ca2+ entry in mammalian cells, our findings with hTRC3a support our previous proposal that TRPCs form capacitative Ca-entry channels.

Amino Acid Sequence↗
PubMed1996

Identification and characterization of a novel type of replication terminator with bidirectional activity on the Bacillus subtilis theta plasmid pLS20.

We have sequenced and analysed a 3.1 kb fragment of the 55 kb endogenous Bacillus subtilis plasmid pLS20 containing its replication functions. Just outside the region required for autonomous replication, a segment of 18 bp was identified as being almost identical to part of the major B. subtilis chromosomal replication terminator. Here, we demonstrate that this segment is part of a functional replication terminator. This newly identified element, designated TerLS20, is the first replication terminator identified on a theta plasmid from a Gram-positive bacterium. TerLS20 is distinct from other known replication terminators in the sense that it is functional in both orientations. The region required for bipolar functionality of TerLS20 was delineated to a sequence of 29 bp, which is characterized by an imperfect dyad symmetry.

Bacillus subtilis↗
PubMed2006

Transient receptor potential channels and caveolin-1: good friends in tight spaces.

Caveolae formation has raised the concept of energy efficiency to new heights. The ultimate purpose of caveolae formation is to colocalize signaling proteins with membrane microdomains in order to facilitate their interaction and improve signal transduction efficiency. Although we know that the main structural protein of caveolae is caveolin, how caveolin interacts with membrane proteins to facilitate their integration into lipid raft domains is unclear. A caveolin-scaffolding domain (CSD) on caveolin itself can associate with membrane proteins such as G proteins and endothelial nitric oxide synthase. In this issue, Kwiatek et al. (p. 1174) report that the TRPC1 channel protein contains a C-terminal CSD-consensus binding sequence that allows for its physical and functional interaction with caveolin-1 in the caveolae of human pulmonary artery endothelial cells (PAEC). Competitive interaction with a CSD-conjugated peptide attenuates thrombin- and thapsigargin-induced Ca2+ influx via store-operated TRPC1 channels. Their data suggest that caveolin-1 can directly regulate TRPC1 function, extending its already ascribed role as a structural protein.

Calcium Channels↗
PubMed2003

Evidence of a role for TRPC channels in VEGF-mediated increased vascular permeability in vivo.

Vascular endothelial growth factor (VEGF) increases vascular permeability by stimulating endothelial Ca(2+) influx. Here we provide evidence that links VEGF-mediated increased permeability and endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) with diacylglycerol (DAG)-mediated activation of the transient receptor potential channels (TRPCs). We used the Landis-Michel technique to measure changes in hydraulic conductivity (L(p)) and fluorescence photometry to quantify changes in endothelial [Ca(2+)](i) in individually perfused Rana mesenteric microvessels in vivo and transfected nonendothelial cells in vitro. The membrane-permeant DAG analog 1-oleoyl-2-acetyl-sn-glycerol (OAG, 100 microM), which is known to increase Ca(2+) influx through TRPCs, transiently increased L(p) 3.8 +/- 1.2-fold (from 1.6 +/- 0.8 to 9.8 +/- 2.7 x 10(-7) cm.s(-1).cmH(2)O(-1); P < 0.0001; n = 18). Protein kinase C inhibition by bisindolylmaleimide (1 microM) did not affect the OAG-induced increases in L(p). OAG also significantly increased microvascular endothelial [Ca(2+)](i) in vivo (n = 13; P < 0.0001), which again was not sensitive to protein kinase C inhibition. VEGF induced a transient increase in endothelial [Ca(2+)](i) in human embryonic kidney cells (HEK-293) that were cotransfected with VEGF receptor 2 and TRPC-6 but not with control, VEGF receptor 2, or TRPC-6 expression vector alone (P < 0.01; n = 9). Flufenamic acid, which has been shown to enhance activity of TRPC-6 but inhibit TRPC-3 and -7, enhanced the VEGF-mediated increase in L(p) in approximately half of the vessels tested but inhibited the response in the other half of the vessels. These data provide evidence consistent with the hypothesis that VEGF increases vascular permeability via DAG-mediated Ca(2+) entry through TRPCs. Although the exact identities of the TRPCs remain to be confirmed, TRPC-6 appears to be a likely candidate in approximately half of the vessels.

Animals↗
PubMed2005

Activation of the endothelial store-operated ISOC Ca2+ channel requires interaction of protein 4.1 with TRPC4.

Store-operated calcium (SOC) entry represents the principal Ca2+ entry pathway into nonexcitable cells. Despite intensive investigation, mechanisms underlying activation of SOC entry have remained elusive. The endothelial ISOC channel is a Ca2+-selective SOC entry channel to which the transient receptor potential (TRP) proteins TRPC1 and TRPC4 contribute subunits. Activation of ISOC is specifically regulated by the spectrin-actin membrane skeleton; however, the nature of coupling between the ISOC channel and membrane skeleton is unknown. Here we demonstrate that protein 4.1 is an essential component of the ISOC channel gating mechanism. Protein 4.1 interacts with TRPC4 and the membrane skeleton. Deletion of the protein 4.1 binding domain on TRPC4 or peptide competition to the protein 4.1 binding domain prevents ISOC activation. These findings reveal that interaction of protein 4.1 with TRPC4 is required for activation of the endothelial ISOC channel.

Binding Sites↗