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PubMed2005

The non-selective cation-permeable channel TRPC3 is a tetrahedron with a cap on the large cytoplasmic end.

TRPC3 plays important roles in neuronal differentiation and immune cell maturation by mediating the cationic current in response to phospholipase C activation, Ca2+ depletion, and diacylglycerol stimulation. Here, we purified the TRPC3 channel using a glycosylated tetramer and observed the structure using electron microscopy. Negatively stained specimens demonstrate homogeneous protein particles containing an internal cavity-like structure. These particle images were picked up by automated pick-up programs, aligned, and classified by the growing neural gas network method. Similarly oriented projections were averaged to decrease the signal-to-noise ratio. The averaged images progress from the top view to the side views, which are representative of their raw images. The top view confirmed the hypothesis of a four-domain structure, and the side view demonstrates a large cytoplasmic domain with a capped structure at the bottom, which is near a predicted locus of ion release. The total image of the protein is a blunt-edged trapezoid of 200 x 200 x 235 A. This large dimension of TRPC3 is also supported by the Stokes radius (92 A) obtained from gel filtration chromatography.

Chromatography, Gel↗
PubMed2002

Comparison of human TRPC3 channels in receptor-activated and store-operated modes. Differential sensitivity to channel blockers suggests fundamental differences in channel composition.

Capacitative calcium entry or store-operated calcium entry in nonexcitable cells is a process whereby the activation of calcium influx across the plasma membrane is signaled by depletion of intracellular calcium stores. Transient receptor potential (TRP) proteins have been proposed as candidates for store-operated calcium channels. Human TRPC3 (hTRPC3), an extensively studied member of the TRP family, is activated through a phospholipase C-dependent mechanism, not by store depletion, when expressed in HEK293 cells. However, store depletion by thapsigargin is sufficient to activate hTRPC3 channels when expressed in DT40 avian B-lymphocytes. To gain further insights into the differences between hTRPC3 channels generated in these two expression systems and further understand the role of hTRPC3 in capacitative calcium entry, we examined the effect of two well characterized inhibitors of capacitative calcium entry, Gd3+ and 2-aminoethoxydiphenyl borane (2APB). We confirmed that in both DT40 cells and HEK293 cells, 1 microm Gd3+ or 30 microm 2APB completely blocked calcium entry due to receptor activation or store depletion. In HEK293 cells, 1 microm Gd3+ did not block receptor-activated hTRPC3-mediated cation entry, whereas 2APB had a partial (approximately 60%) inhibitory effect. Interestingly, store-operated hTRPC3-mediated cation entry in DT40 cells was also partially inhibited by 2APB, whereas 1 microm Gd3+ completely blocked store-operated hTRPC3 activity in these cells. Furthermore, the sensitivity of store-operated hTRPC3 channels to Gd3+ in DT40 cells was similar to the endogenous store-operated channels, with essentially 100% block of activity at concentrations as low as 0.1 microm. Finally, Gd3+ has a rapid inhibitory effect when added to fully developed hTRPC3-mediated calcium entry, suggesting a direct action of Gd3+ on hTRPC3 channels. The distinct action of these inhibitors on hTRPC3-mediated cation entry in these two cell types may result from their different modes of activation and may also reflect differences in basic channel structure.

Boron Compounds↗
PubMed2005

Expression of a truncated form of inositol 1,4,5-trisphosphate receptor type III in the cytosol of DT40 triple inositol 1,4,5-trisphosphate receptor-knockout cells.

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel playing a major role in Ca2+ signaling. Three isoforms of IP3R have been identified and most cell types express different proportions of each isoform. The DT40 B lymphocyte cell line lacking all three IP3R isoforms (DT40IP3R-KO cells) represents an excellent model to re-express any recombinant IP3R and analyze its specific properties. In the study presented here, we confirmed that DT40IP3R-KO cells do not express any IP3-sensitive Ca2+ release channel. However, with an immunoblot approach and a [3H]IP3 binding approach we demonstrated the presence of a C-terminally truncated form of IP3R type III in the cytosolic fraction of DT40IP3R-KO cells. We further showed that this truncated IP3R retained the ability to couple to the Ca2+ entry channel TRPC6. Therefore, a word of caution is offered about the interpretation of results obtained in using DT40IP3R-KO cells to study the cellular mechanisms of Ca2+ entry.

Animals↗
PubMed2005

TRP channels in mechanosensation.

Channels of the TRP superfamily have sensory roles in a wide variety of receptor cells, especially in mechanosensation. In some cases, the channels appear to be directly activated by mechanical force; in others, they appear to be downstream of a messenger pathway initiated by force on a non-channel sensor. A remaining challenge for most of these mechanosensory TRPs is to clarify the specific mechanism of activation.

Animals↗
PubMed2005

Generation of functional ion-channel tools by E3 targeting.

Here we describe a strategy for generating ion-channel inhibitors. It takes advantage of antibody specificity combined with a pattern recognition approach that targets the third extracellular region (E3) of a channel. To test the concept, we first focused on TRPC5, a member of the transient receptor potential (TRP) calcium channel family, the study of which has been hindered by poor pharmacological tools. Extracellular application of E3-targeted anti-TRPC5 antibody led to a specific TRPC5 inhibitor, enabling TRPC5 to be distinguished from its closest family members, and TRPC5 function to be explored in a relatively intractable physiological system. E3 targeting was further applied to voltage-gated sodium channels, leading to discovery of a subtype-specific inhibitor of Na(V)1.5. These examples illustrate the potential power of E3 targeting as a systematic method for producing gene-type specific ion-channel inhibitors for use in routine assays on cells or tissues from a range of species and having therapeutic potential.

Antibodies, Monoclonal↗
PubMed2004

Insights into the molecular nature of magnesium homeostasis.

Magnesium is an important cofactor for many biological processes, such as protein synthesis, nucleic acid stability, or neuromuscular excitability. Extracellular magnesium concentration is tightly regulated by the extent of intestinal absorption and renal excretion. Despite the critical role of magnesium handling, the exact mechanisms mediating transepithelial transport remained obscure. In the past few years, the genetic disclosure of inborn errors of magnesium handling revealed several new proteins along with already known molecules unexpectedly involved in renal epithelial magnesium transport, e.g., paracellin-1, a key player in paracellular magnesium and calcium reabsorption in the thick ascending limb or the gamma-subunit of the Na(+)-K(+)-ATPase in the distal convoluted tubule. In this review, we focus on TRPM6, an ion channel of the "transient receptor potential (TRP) gene family, which, when mutated, causes a combined defect of intestinal magnesium absorption and renal magnesium conservation as observed in primary hypomagnesemia with secondary hypocalcemia.

Animals↗
PubMed2004

Plasma membrane localization of TRPC channels: role of caveolar lipid rafts.

GPCR-mediated activation of the Ca2+ signalling cascade leads to stimulation of Ca2+ influx into non-excitable cells. Both store-dependent and independent channels likely contribute towards this Ca2+ influx. However, the identity of the channels and exact mechanism by which they are activated remains elusive. The TRPC family of proteins has been proposed as molecular components of these channels. Studies from our laboratory and others have shown that mammalian TRPC proteins are assembled in a multiprotein complex that includes various key Ca2+ signalling proteins. However, relatively little is known regarding the mechanisms involved in the assembly of the TRPC channel complex in the plasma membrane. We have reported that TRPC1 and TRPC3 signalling complexes are associated with caveolar lipid raft domains (LRDs) in the plasma membrane. Recently we have examined the role of caveolin-1 in the regulation of TRPC channels and store-operated Ca2+ entry (SOCE). Based on our studies, we suggest that (1) caveolin 1 has a potentially critical role in the localization of TRPC channels plasma membrane caveolar LRDs, and (2) the molecular architecture of caveolae can facilitate intramolecular interactions between TRPC channels and associated proteins that are involved in activation and/or inactivation of SOCE.

Animals↗
PubMed2005

Functional characterization and physiological relevance of the TRPC3/6/7 subfamily of cation channels.

The mammalian transient receptor potential (TRP) superfamily of cation channels can be divided into six major families. Among them, the "classical" or "canonical" TRPC family is most closely related to Drosophila TRP, the founding member of the superfamily. All seven channels of this family designated TRPC1-7 share the common property of activation through phospholipase C (PLC)-coupled receptors, but their gating by receptor- or store-operated mechanisms is still controversial. The TRPC3, 6, and 7 channels are 75% identical and are also gated by direct exposure to diacylglycerols (DAG). TRPC3, 6, and 7 interact physically and, upon coexpression, coassemble to form functional tetrameric channels. This review will focus on the TRPC3/6/7 subfamily and describe their functional properties and regulation as homomers obtained from overexpression studies in cell lines. It will also summarize their heteromultimerization potential in vitro and in vivo and will present preliminary data concerning their physiological functions analyzed in isolated tissues with downregulated channel activity and gene-deficient mouse models.

Animals↗
PubMed2003

Regulation of canonical transient receptor potential (TRPC) channel function by diacylglycerol and protein kinase C.

The mechanism of receptor-induced activation of the ubiquitously expressed family of mammalian canonical transient receptor potential (TRPC) channels has been the focus of intense study. Primarily responding to phospholipase C (PLC)-coupled receptors, the channels are reported to receive modulatory input from diacylglycerol, endoplasmic reticulum inositol 1,4,5-trisphosphate receptors and Ca2+ stores. Analysis of TRPC5 channels transfected within DT40 B cells and deletion mutants thereof revealed efficient activation in response to PLC-beta or PLC-gamma activation, which was independent of inositol 1,4,5-trisphoshate receptors or the content of stores. In both HEK293 cells and DT40 cells, TRPC5 and TRPC3 channel responses to PLC activation were highly analogous, but only TRPC3 and not TRPC5 channels responded to the addition of the permeant diacylglycerol (DAG) analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG). However, OAG application or elevated endogenous DAG, resulting from either DAG lipase or DAG kinase inhibition, completely prevented TRPC5 or TRPC4 activation. This inhibitory action of DAG on TRPC5 and TRPC4 channels was clearly mediated by protein kinase C (PKC), in distinction to the stimulatory action of DAG on TRPC3, which is established to be PKC-independent. PKC activation totally blocked TRPC3 channel activation in response to OAG, and the activation was restored by PKC-blockade. PKC inhibition resulted in decreased TRPC3 channel deactivation. Store-operated Ca2+ entry in response to PLC-coupled receptor activation was substantially reduced by OAG or DAG-lipase inhibition in a PKC-dependent manner. However, store-operated Ca2+ entry in response to the pump blocker, thapsigargin, was unaffected by PKC. The results reveal that each TRPC subtype is strongly inhibited by DAG-induced PKC activation, reflecting a likely universal feedback control on TRPCs, and that DAG-mediated PKC-independent activation of TRPC channels is highly subtype-specific. The profound yet distinct control by PKC and DAG of the activation of TRPC channel subtypes is likely the basis of a spectrum of regulatory phenotypes of expressed TRPC channels.

Animals↗
PubMed2004

A TRPC1/TRPC3-mediated increase in store-operated calcium entry is required for differentiation of H19-7 hippocampal neuronal cells.

Store-operated calcium entry (SOCE) and TRPC protein expression were investigated in the rat-derived hippocampal H19-7 cell line. Thapsigargin-stimulated Ba2+ entry and the expression of TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7 mRNA and protein were observed in proliferating H19-7 cells. When cells were placed under differentiating conditions, a change in TRPC homolog expression profile occurred. The expression of TRPC1 and TRPC3 mRNA and protein dramatically increased, while the expression of TRPC4 and TRPC7 mRNA and protein dramatically decreased; in parallel a 3.4-fold increase in the level of thapsigargin-stimulated Ba2+ entry was observed and found to be inhibited by 2-aminoethoxydiphenylborane. The selective suppression of TRPC protein levels by small interfering RNA (siRNA) approaches indicated that TRPC1 and TRPC3 are involved in mediating SOCE in proliferating H19-7 cells. Although TRPC4 and TRPC7 are expressed at much higher levels than TRPC1 and TRPC3 in proliferating cells, they do not appear to mediate SOCE. The co-expression of siRNA specific for TRPC1 and TRPC3 in proliferating cells inhibited approximately the same amount of SOCE as observed with expression of either siRNA alone, suggesting that TRPC1 and TRPC3 work in tandem to mediate SOCE. Under differentiating conditions, co-expression of siRNA for TRPC1 and TRPC3 blocked the normal 3.4-fold increase in SOCE and in turn blocked the differentiation of H19-7 cells. This study suggests that placing H19-7 cells under differentiating conditions significantly alters TRPC gene expression and increases the level of SOCE and that this increase in SOCE is necessary for cell differentiation.

Animals↗
PubMed2002

TRPC6 is a candidate channel involved in receptor-stimulated cation currents in A7r5 smooth muscle cells.

To investigate the possible role of members of the mammalian transient receptor potential (TRP) channel family (TRPC1-7) in vasoconstrictor-induced Ca(2+) entry in vascular smooth muscle cells, we studied [Arg(8)]-vasopressin (AVP)-activated channels in A7r5 aortic smooth muscle cells. AVP induced an increase in free cytosolic Ca(2+) concentration ([Ca(2+)](i)) consisting of Ca(2+) release and Ca(2+) influx. Whole cell recordings revealed the activation of a nonselective cation current with a doubly rectifying current-voltage relation strikingly similar to those described for some heterologously expressed TRPC isoforms. The current was also stimulated by direct activation of G proteins as well as by activation of the phospholipase Cgamma-coupled platelet-derived growth factor receptor. Currents were not activated by store depletion or increased [Ca(2+)](i). Application of 1-oleoyl-2-acetyl-sn-glycerol stimulated the current independently of protein kinase C, a characteristic property of the TRPC3/6/7 subfamily. Like TRPC6-mediated currents, cation currents in A7r5 cells were increased by flufenamate. Northern hybridization revealed mRNA coding for TRPC1 and TRPC6. We therefore suggest that TRPC6 is a molecular component of receptor-stimulated Ca(2+)-permeable cation channels in A7r5 smooth muscle cells.

Animals↗
PubMed2001

Physiology, phylogeny, and functions of the TRP superfamily of cation channels.

The transient receptor potential (TRP) protein superfamily consists of a diverse group of Ca(2+) permeable nonselective cation channels that bear structural similarities to Drosophila TRP. TRP-related proteins play important roles in nonexcitable cells, as demonstrated by the recent finding that a mammalian TRPC protein is expressed in endothelial cells and functions in vasorelaxation. However, an emerging theme is that many TRP-related proteins are expressed predominantly in the nervous system and function in sensory physiology. The TRP superfamily can be divided into six subfamilies, the first of which is composed of the "classical TRPs" (TRPC subfamily). These proteins all share the common features of three to four ankryin repeats, >/=30% amino acid homology over >/=750 amino acids, and a gating mechanism that operates through phospholipase C. Some classical TRPs may be store-operated channels (SOCs), which are activated by release of Ca(2+) from internal stores. The mammalian TRPC proteins are also expressed in the central nervous system, and several are highly enriched in the brain. One TRPC protein has been implicated in the pheromone response. The archetypal TRP, Drosophila TRP, is predominantly expressed in the visual system and is required for phototransduction. Many members of a second subfamily (TRPV) function in sensory physiology. These include VR1 and OSM-9, which respond to heat, osmolarity, odorants, and mechanical stimuli. A third subfamily, TRPN, includes proteins with many ankyrin repeats, one of which, NOMPC, participates in mechanotransduction. Among the members of a fourth subfamily, TRPM, is a putative tumor suppressor termed melastatin, and a bifunctional protein, TRP-PLIK, consisting of a TRPM channel fused to a protein kinase. PKD2 and mucolipidin are the founding members of the TRPP and TRPML subfamilies, respectively. Mutations in PKD2 are responsible for polycystic kidney disease, and mutations in mucolipidin result in a severe neurodegenerative disorder. Recent studies suggest that alterations in the activities of SOC and TRP channels may be at the heart of several additional neurodegenerative diseases. Thus, TRP channels may prove to be important new targets for drug discovery.

Animals↗
PubMed2004

Multiple regulation by calcium of murine homologues of transient receptor potential proteins TRPC6 and TRPC7 expressed in HEK293 cells.

We investigated, by using the patch clamp technique, Ca2+-mediated regulation of heterologously expressed TRPC6 and TRPC7 proteins in HEK293 cells, two closely related homologues of the transient receptor potential (TRP) family and molecular candidates for native receptor-operated Ca2+ entry channels. With nystatin-perforated recording, the magnitude and time courses of activation and inactivation of carbachol (CCh; 100 microM)-activated TRPC6 currents (I(TRPC6)) were enhanced and accelerated, respectively, by extracellular Ca2+ (Ca2+(o)) whether it was continuously present or applied after receptor stimulation. In contrast, Ca2+(o) solely inhibited TRPC7 currents (I(TRPC7)). Vigorous buffering of intracellular Ca2+ (Ca2+(i)) under conventional whole-cell clamp abolished the slow potentiating (i.e. accelerated activation) and inactivating effects of Ca2+(o), disclosing fast potentiation (EC50: approximately 0.4 mM) and inhibition (IC50: approximately 4 mM) of I(TRPC6) and fast inhibition (IC50: approximately 0.4 mM) of I(TRPC7). This inhibition of I(TRPC6) and I(TRPC7) seems to be associated with voltage-dependent reductions of unitary conductance and open probability at the single channel level, whereas the potentiation of I(TRPC6) showed little voltage dependence and was mimicked by Sr2+ but not Ba2+. The activation process of I(TRPC6) or its acceleration by Ca2+(o) probably involves phosphorylation by calmodulin (CaM)-dependent kinase II (CaMKII), as pretreatment with calmidazolium (3 microM), coexpression of Ca2+-insensitive mutant CaM, and intracellular perfusion of the non-hydrolysable ATP analogue AMP-PNP and a CaMKII-specific inhibitory peptide all effectively prevented channel activation. However, this was not observed for TRPC7. Instead, single CCh-activated TRPC7 channel activity was concentration-dependently suppressed by nanomolar Ca2+(i) via CaM and conversely enhanced by IP3. In addition, the inactivation time course of I(TRPC6) was significantly retarded by pharmacological inhibition of protein kinase C (PKC). These results collectively suggest that TRPC6 and 7 channels are multiply regulated by Ca2+ from both sides of the membrane through differential Ca2+-CaM-dependent and -independent mechanisms.

Animals↗
PubMed2005

Functional role of TRPC proteins in native systems: implications from knockout and knock-down studies.

Available data on transient receptor potential channel (TRPC) protein functions indicate that these proteins represent essential constituents of agonist-activated and phospholipase C-dependent cation entry pathways in primary cells which contribute to the elevation of cytosolic Ca2+. In addition, a striking number of biological functions have already been assigned to the various TRPC proteins, including mechanosensing activity (TRPC1), chemotropic axon guidance (TRPC1 and TRPC3), pheromone sensing and the regulation of sexual and social behaviour (TRPC2), endothelial-dependent regulation of vascular tone, endothelial permeability and neurotransmitter release (TRPC4), axonal growth (TRPC5), modulation of smooth muscle tone in blood vessels and lung and regulation of podocyte structure and function in the kidney (TRPC6). The lack of compounds which specifically block or activate TRPC proteins impairs the analysis of TRPC function in primary cells. We therefore concentrate in this contribution on (i) studies of TRPC-deficient mouse lines, (ii) data obtained by gene-silencing approaches using antisense oligonucleotides or RNA interference, (iii) expression experiments employing dominant negative TRPC constructs, and (iv) recent data correlating mutations of TRPC genes associated with human disease.

Animals↗
PubMed2005

Essential role of a Ca2+-selective, store-operated current (ISOC) in endothelial cell permeability: determinants of the vascular leak site.

Store-operated calcium (SOC) entry is sufficient to disrupt the extra-alveolar, but not the alveolar, endothelial cell barrier. Mechanism(s) underlying such insensitivity to transitions in cytosolic calcium ([Ca2+]i) in microvascular endothelial cells are unknown. Depletion of stored Ca2+ activates a larger SOC entry response in extra-alveolar (pulmonary artery; PAECs) than alveolar (pulmonary microvascular; PMVECs) endothelial cells. In vivo permeation studies revealed that Ca2+ store depletion activates similar nonselective cationic conductances in PAECs and PMVECs, while only PAECs possess the calcium-selective, store-operated Ca2+ entry current, I(SOC). Pretreatment with the type 4 phosphodiesterase inhibitor, rolipram, abolished thapsigargin-activated I(SOC) in PAECs, and revealed I(SOC) in PMVECs. Rolipram pretreatment shifted the thapsigargin-induced fluid leak site from extra-alveolar to alveolar vessels in the intact pulmonary circulation. Thus, our results indicate I(SOC) provides a [Ca2+]i source that is needed to disrupt the endothelial cell barrier, and demonstrate that intracellular events controlling I(SOC) activation coordinate the site-specific vascular response to inflammation.

Adenylyl Cyclases↗
PubMed2007

TRPC3: a multifunctional, pore-forming signalling molecule.

TRPC3 represents one of the first identified mammalian relatives of the Drosophila trp gene product. Despite intensive biochemical and biophysical characterization as well as numerous attempts to uncover its physiological role in native cell systems, this channel protein still represents one of the most enigmatic members of the transient receptor potential (TRP) superfamily. TRPC3 is significantly expressed in brain and heart and likely to play a role in both non-excitable as well as excitable cells, being potentially involved in a wide spectrum of Ca2+ signalling mechanisms. Its ability to associate with a variety of partner proteins apparently enables TRPC3 to form different cation channels in native cells. TRPC3 cation channels display unique gating and regulatory properties that allow for recognition and integration of multiple input stimuli including lipid mediators and cellular Ca2+ gradients as well as redox signals. The physiological/pathophysiological functions of this highly versatile cation channel protein are as yet barely delineated. Here we summarize current knowledge on properties and possible signalling functions of TRPC3 and discuss the potential biological relevance of this signalling molecule.

Animals↗
PubMedDate not supplied

The pore of TRP channels: trivial or neglected?

Ultimate proof that a protein contributes to the pore of an ion channel is demonstrating that pore properties can be altered by mutations to the putative pore-forming region. To date this has only been achieved for a few TRP proteins, and only within the TRPV subfamily. The location and structure of the pore region and selectivity filter of most TRP proteins, including all members of the TRPM and TRPC subfamilies, is currently unknown. In this review we give a short overview of the limited current knowledge about TRP channel pores and argue that further study is needed, not only for a better understanding of the mechanisms underlying cation permeation, but also to establish that all members of the TRP superfamily indeed function as bona fide ion channels.

Amino Acid Sequence↗