Structural mechanism of human TRPC3 and TRPC6 channel regulation by their intracellular calcium-binding sites

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Receptor-mediated regulation of the nonselective cation channels TRPC4 and TRPC5

Mammalian transient receptor potential channels (TRPCs) form a family of Ca(2+)-permeable cation channels currently consisting of seven members, TRPC1-TRPC7. These channels have been proposed to be molecular correlates for capacitative Ca(2+) entry channels. There are only a few studies on the regulation and properties of the subfamily consisting of TRPC4 and TRPC5, and there are contradictory reports concerning the possible role of intracellular Ca(2+) store depletion in channel activation. We therefore investigated the regulatory and biophysical properties of murine TRPC4 and TRPC5 (mTRPC4/5) heterologously expressed in human embryonic kidney cells. Activation of G(q/11)-coupled receptors or receptor tyrosine kinases induced Mn(2+) entry in fura-2-loaded mTRPC4/5-expressing cells. Accordingly, in whole-cell recordings, stimulation of G(q/11)-coupled receptors evoked large, nonselective cation currents, an effect mimicked by infusion of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS). However, depletion of intracellular Ca(2+) stores failed to activate mTRPC4/5. In inside-out patches, single channels with conductances of 42 and 66 picosiemens at -60 mV for mTRPC4 and mTRPC5, respectively, were stimulated by GTPgammaS in a membrane-confined manner. Thus, mTRPC4 and mTRPC5 form nonselective cation channels that integrate signaling pathways from G-protein-coupled receptors and receptor tyrosine kinases independently of store depletion. Furthermore, the biophysical properties of mTRPC4/5 are inconsistent with those of I(CRAC), the most extensively characterized store-operated current.     

Trafficking mechanisms and regulation of TRPC channels

TRPC channels are Ca²⁺-permeable cation channels which are regulated downstream from receptor-coupled PIP₂ hydrolysis. These channels contribute to a wide variety of cellular functions. Loss or gain of channel function has been associated with dysfunction and aberrant physiology. TRPC channel functions are influenced by their physical and functional interactions with numerous proteins that determine their regulation, scaffolding, trafficking, as well as their effects on the downstream cellular processes. Such interactions also compartmentalize the Ca²⁺ signals arising from TRPC channels. A large number of studies demonstrate that trafficking is a critical mode by which plasma membrane localization and surface expression of TRPC channels are regulated. This review will provide an overview of intracellular trafficking pathways as well as discuss the current state of knowledge regarding the mechanisms and components involved in trafficking of the seven members of the TRPC family (TRPC1–TRPC7).     

TRPC6 participates in the regulation of cytosolic basal calcium concentration in murine resting platelets

Cytosolic-free Ca^2 + plays a crucial role in blood platelet function and is essential for thrombosis and hemostasis. Therefore, cytosolic-free Ca^2 + concentration is tightly regulated in this cell. TRPC6 is expressed in platelets, and an important role for this Ca^2 + channel in Ca^2 + homeostasis has been reported in other cell types. The aim of this work is to study the function of TRPC6 in platelet Ca^2 + homeostasis. The absence of TRPC6 resulted in an 18.73% decreased basal [Ca^2 +]_c in resting platelets as compared to control cells. Further analysis confirmed a similar Ca^2 + accumulation in wild-type and TRPC6-deficient mice; however, passive Ca^2 + leak rates from agonist-sensitive intracellular stores were significantly decreased in TRPC6-deficient platelets. Biotinylation studies indicated the presence of an intracellular TRPC6 population, and subcellular fractionation indicated their presence on endoplasmic reticulum membranes. Moreover, the presence of intracellular calcium release in platelets stimulated with 1-oleoyl-2-acetyl-sn-glycerol further suggested a functional TRPC6 population located on the intracellular membranes surrounding calcium stores. However, coimmunoprecipitation assay confirmed the absence of STIM1–TRPC6 interactions in resting conditions. This findings together with the absence of extracellular Mn^2 + entry in resting wild-type platelets indicate that the plasma membrane TRPC6 fraction does not play a significant role in the maintenance of basal [Ca^2 +]_c in mouse platelets. Our results suggest an active participation of the intracellular TRPC6 fraction as a regulator of basal [Ca^2 +]_c, controlling the passive Ca^2 + leak rate from agonist-sensitive intracellular Ca^2 + stores in resting platelets.     

TRPC channel lipid specificity and mechanisms of lipid regulation

TRPC channels are a subset of the transient receptor potential (TRP) proteins widely expressed in mammalian cells. They are thought to be primarily involved in determining calcium or sodium entry and have broad-ranging functions that include regulation of cell proliferation, motility and contraction. The channels do not respond to a single stimulator but rather are activated or modulated by a multiplicity of factors, potentially existing as integrators at the plasma membrane. This review considers the sensitivity of TRPCs to lipid factors, with focus on sensitivities to diacylglycerols, lysophospholipids, arachidonic acid and its metabolites, sphingosine-1-phosphate (S1P), cholesterol and derivatives, and other lipid factors such as gangliosides. Promiscuous and selective lipid-sensing are apparent. In many cases the lipids stimulate channel function or increase insertion of channels in the membrane. Both direct and indirect (receptor-dependent) lipid effects are evident. Although information is limited, the lipid profiles are consistent with TRPCs having close working relationships with phospholipase C and A2 enzymes. We need much more information about lipid-sensing by TRPCs if we are to fully appreciate its significance, but the available data suggest that lipid-sensing is a key, but not exclusive, aspect of TRPC biology.     

Complex regulation of the TRPC3, 6 and 7 channel subfamily by diacylglycerol and phosphatidylinositol-4,5-bisphosphate

TRPC3, 6 and 7 channels constitute a subgroup of non-selective, calcium-permeable cation channels within the TRP superfamily that are activated by products of phospholipase C-mediated breakdown of phosphatidylinositol-4,5-bisphosphate (PIP(2)). A number of ion channels, including other members of the TRP superfamily, are regulated directly by PIP(2). However, there is little information on the regulation of the TRPC channel subfamily by PIP(2). Pretreatment of TRPC7-expressing cells with a drug that blocks the synthesis of polyphosphoinositides inhibited the ability of the synthetic diacylglycerol, oleyl-acetyl glycerol, to activate TRPC7. In excised patches, TRPC7 channels were robustly activated by application of PIP(2) or ATP, but not by inositol 1,4,5-trisphosphate. Similar results were obtained with TRPC6 and TRPC3, although the effects of PIP(2) were somewhat less and with TRPC3 there was no significant effect of ATP. In the cell-attached configuration, TRPC7 channels could be activated by the synthetic diacylglycerol analog, oleyl-acetyl glycerol. However, this lipid mediator did not activate TRPC7 channels in excised patches. In addition, channel activation by PIP(2) in excised patches was significantly greater than that observed with oleyl-acetyl glycerol in the cell-attached configuration. These findings reveal complex regulation of TRPC channels by lipid mediators. The results also reveal for the first time direct activation by PIP(2) of members of the TRPC ion channel subfamily.     

Mechanisms of lipid regulation and lipid gating in TRPC channels

TRPC proteins form cation channels that integrate and relay cellular signals by mechanisms involving lipid recognition and lipid-dependent gating. The lipohilic/amphiphilic molecules that function as cellular activators or modulators of TRPC proteins span a wide range of chemical structures. In this context, cellular redox balance is likely linked to the lipid recognition/gating features of TRPC channels. Both classical ligand-protein interactions as well as indirect and promiscuous sensory mechanisms have been proposed. Some of the recognition processes are suggested to involve ancillary lipid-binding scaffolds or regulators as well as dynamic protein–protein interactions determined by bilayer architecture. A complex interplay of protein–protein and protein-lipid interactions is likely to govern the gating and/or plasma membrane recruitment of TRPC channels, thereby providing a distinguished platform for signal integration and coincident signal detection. Both the primary molecular event(s) of lipid recognition by TRPC channels as well as the transformation of these events into distinct gating movements is poorly understood at the molecular level, and it remains elusive whether lipid sensing in TRPCs is conferred to a distinct sensor domain. Recent structural information on the molecular action of lipophilic activators in distantly related members of the TRP superfamily encourages speculations on TRPC gating mechanisms involved in lipid recognition/gating. This review aims to provide an update on the current understanding of the lipid–dependent control of TRPC channels with focus on the TRPC lipid sensing, signal-integration hub and a short discussion of potential links to redox signaling.     

Store-operated Ca2+ channels formed by TRPC1, TRPC6 and Orai1 and non-store-operated channels formed by TRPC3 are involved in the regulation of NADPH oxidase in HL-60 granulocytes

Ca(2+) influx has been shown to be essential for NADPH oxidase activity which is involved in the inflammatory process. Ca(2+) conditions underlying the oxidative response are clearly delineated. Here, we show that store-operated Ca(2+) entry (SOCE) is required at the beginning of NADPH oxidase activation in response to fMLF (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) in neutrophil-like HL-60 cells. When extracellular Ca(2+) is initially removed, early addition of Ca(2+) after stimulation causes a complete restoration of Ca(2+) entry and H(2)O(2) production. Both Ca(2+) entry and H(2)O(2) production are decreased by purported SOCE blockers, 2-aminoethoxydiphenyl borane (2-APB) and SK&F 96365. Endogenously expressed TRPC (transient receptor potential canonical) homologues and Orai1 were investigated for their role in supporting store-operated Ca(2+) channels activity. TRPC1, TRPC6 and Orai1 knock-out by siRNA resulted in the inhibition of Ca(2+) influx and H(2)O(2) production in response to fMLF and thapsigargin while suppression of TRPC3 had no effect on thapsigargin induced-SOCE. 2-APB and SK&F 96365 were able to amplify the reduction of fMLF-stimulated Ca(2+) entry and H(2)O(2) production observed in cells transfected by TRPC3 siRNA. In summary, Ca(2+) influx in HL-60 cells relies on different membrane TRPC channels and Orai1 for allowing NADPH oxidase activation. TRPC3 primarily mediates SOCE-independent pathways and TRPC1, TRPC6 and Orai1 exclusively contribute to SOCE.     

TRPC6在中枢神经系统的作用

TRPC6（Thetransientreceptorpotentialcanonical6）为瞬时受体电位（TRP）超家族的成员之一,编码钙可通透的非选择性阳离子通道。其具有六次跨膜结构。TRPC6同型或异型四聚体通道由TRPC6蛋白相互结合形成或与同在一个亚家族的TRPC3,TRPC7形成。TRPC6通道可被G蛋白耦联受体（GPCR）和受体酪氨酸激酶（receptortyrosinekinasesRTK）通过激活磷脂酶C（PLC）激活。其还可直接被第二信使DAG（diacylglycer01）激活。已有研究证实该通道通过激活上述信号传导通路参与了多种生理过程。TRPC6基因编码的蛋白在人体多个部位均有表达。TRPC6在中枢神经系统广泛表达。其在不同部位的表达量不同,并与TRPC家族的其他成员一起参与了多种生理过程。TRPC6引起的细胞阳离子浓度的变化可能参与了多种神经系统疾病的发生发展过程。因此。研究TRPC6在中枢神经系统中的作用对疾病发病机制的了解及治疗变得更有意义。本文就TRPC6在中枢神经系统中的作用进行综述,并主要介绍其在树突发育,神经元保护及细胞生长方面的作用。     

TRPC6 在中枢神经系统的作用

摘要：TRPC6(The transient receptor potential canonical 6)为瞬时受体电位(TRP)超家族的成员之一,编码钙可通透的非选择性阳离 子通道。其具有六次跨膜结构。TRPC6 同型或异型四聚体通道由TRPC6 蛋白相互结合形成或与同在一个亚家族的TRPC3, TRPC7 形成。TRPC6 通道可被G 蛋白耦联受体（GPCR）和受体酪氨酸激酶（receptor tyrosine kinases RTK）通过激活磷脂酶C (PLC)激活。其还可直接被第二信使DAG (diacylglycerol)激活。已有研究证实该通道通过激活上述信号传导通路参与了多种生理 过程。TRPC6 基因编码的蛋白在人体多个部位均有表达。TRPC6 在中枢神经系统广泛表达。其在不同部位的表达量不同,并与 TRPC家族的其他成员一起参与了多种生理过程。TRPC6 引起的细胞阳离子浓度的变化可能参与了多种神经系统疾病的发生发 展过程。因此,研究TRPC6 在中枢神经系统中的作用对疾病发病机制的了解及治疗变得更有意义。本文就TRPC6 在中枢神经系 统中的作用进行综述,并主要介绍其在树突发育,神经元保护及细胞生长方面的作用。     

Calmodulin and calcium interplay in the modulation of TRPC5 channel activity. Identification of a novel C-terminal domain for calcium/calmodulin-mediated facilitation

TRPC5 forms Ca2+-permeable nonselective cation channels important for neurite outgrowth and growth cone morphology of hippocampal neurons. Here we studied the activation of mouse TRPC5 expressed in Chinese hamster ovary and human embryonic kidney 293 cells by agonist stimulation of several receptors that couple to the phosphoinositide signaling cascade and the role of calmodulin (CaM) on the activation. We showed that exogenous application of 10 microM CaM through patch pipette accelerated the agonist-induced channel activation by 2.8-fold, with the time constant for half-activation reduced from 4.25 +/- 0.4 to 1.56 +/- 0.85 min. We identified a novel CaM-binding site located at the C terminus of TRPC5, 95 amino acids downstream from the previously determined common CaM/IP3R-binding (CIRB) domain for all TRPC proteins. Deletion of the novel CaM-binding site attenuated the acceleration in channel activation induced by CaM. However, disruption of the CIRB domain from TRPC5 rendered the channel irresponsive to agonist stimulation without affecting the cell surface expression of the channel protein. Furthermore, we showed that high (>5 microM) intracellular free Ca2+ inhibited the current density without affecting the time course of TRPC5 activation by receptor agonists. These results demonstrated that intracellular Ca2+ has dual and opposite effects on the activation of TRPC5. The novel CaM-binding site is important for the Ca2+/CaM-mediated facilitation, whereas the CIRB domain is critical for the overall response of receptor-induced TRPC5 channel activation.     

N-linked protein glycosylation is a major determinant for basal TRPC3 and TRPC6 channel activity

The TRPC family of receptor-activated cation channels (TRPC channels) can be subdivided into four subfamilies based on sequence homology as well as functional similarities. Members of the TRPC3/6/7 subfamily share common biophysical characteristics and are activated by diacylglycerol in a membrane-delimited manner. At present, it is only poorly understood whether members of the TRPC3/6/7 subfamily are functionally redundant or whether they serve distinct cellular roles. By electrophysiological and fluorescence imaging strategies we show that TRPC3 displays considerable constitutive activity, while TRPC6 is a tightly regulated channel. To identify potential molecular correlates accounting for the functional difference, we analyzed the glycosylation pattern of TRPC6 compared with TRPC3. Two NX(S/T) motifs in TRPC6 were mutated (Asn to Gln) by in vitro mutagenesis to delete one or both extracellular N-linked glycosylation sites. Immunoblotting analysis of HEK 293 cell lysates expressing TRPC6 wild type and mutants favors a model of TRPC6 that is dually glycosylated within the first (e1) and second extracellular loop (e2) as opposed to the monoglycosylated TRPC3 channel (Vannier, B., Zhu, X., Brown, D., and Birnbaumer, L. (1998) J. Biol. Chem. 273, 8675-8679). Elimination of the e2 glycosylation site, missing in the monoglycosylated TRPC3, was sufficient to convert the tightly receptor-regulated TRPC6 into a constitutively active channel, displaying functional characteristics of TRPC3. Reciprocally, engineering of an additional second glycosylated site in TRPC3 to mimic the glycosylation status in TRPC6 markedly reduced TRPC3 basal activity. We conclude that the glycosylation pattern plays a pivotal role for the tight regulation of TRPC6 through phospholipase C-activating receptors.     

TRPC6 and FSGS: the latest TRP channelopathy

Focal and segmental glomerulosclerosis (FSGS) is a common cause of nephrotic syndrome in children and adults throughout the world. In the past 50 years, significant advances have been made in the identification and characterization of familial forms of nephrotic syndrome and FSGS. Resultant to these pursuits, several podocyte structural proteins such as nephrin, podocin, alpha-actinin 4 (ACTN4), and CD2-associated protein (CD2AP) have emerged to provide critical insight into the pathogenesis of hereditary nephrotic syndromes. The latest advance in familial FSGS has been the discovery of a mutant form of canonical transient receptor potential cation channel 6 (TRPC6), which causes an increase in calcium transients and essentially a gain of function in this cation channel located on the podocyte cell membrane. The TRP ion channel family is a diverse group of cation channels united by a common primary structure which contains six membrane-spanning domains, with both carboxy and amino termini located intracellularly. TRP channels are unique in their ability to activate independently of membrane depolarization. TRPC6 channels have been shown to be activated via phospholipase C stimulation. The mechanisms by which mutant TRPC6 causes an increase in intracellular calcium and leads to glomerulosclerosis are unknown. Mutant TRPC6 may affect critical interactions with the aforementioned podocyte structural proteins, leading to abnormalities in the slit diaphragm or podocyte foot processes. Mutant TRPC6 may also amplify injurious signals mediated by Ang II, a common final pathway of podocyte apoptosis in various mammalian species. Current evidence also suggests that blocking TRPC6 channels may be of therapeutic benefit in idiopathic FSGS, a disease with a generally poor prognosis. Preliminary experiments reveal the commonly used immunosuppressive agent FK-506 can inhibit TRPC6 activity in vivo. This creates the exciting possibility that blocking TRPC6 channels within the podocyte may translate into long-lasting clinical benefits in patients with FSGS.     

Calmodulin and calmodulin-mediated processes in plants

Abstract. The Ca²⁺ -binding protein calmodulin is found in all plants investigated so far. The comparison of the biochemical and functional properties reveals that it is structurally conserved and functionally preserved throughout the plant and animal kingdom. Among the plant enzymes so far known to be dependent on the Ca²⁺ -calmodulin complex are NAD kinase(s), Ca²⁺ -transport ATPase, quinate: NAD⁺ oxidoreductase, soluble and membrane bound protein kinases, and H⁺ -transport ATPase. Calmodulin may play also an important role in the regulation of other cellular reactions, such as hormone-mediated processes, secretion of enzymes, and contractile mechanisms. On the basis of the NAD kinase and its regulation by light and Ca²⁺ -calmodulin, it is suggested that changes in the cellular, free Ca²⁺ concentration following stimulation may alter the metabolism of a plant cell. According to this suggestion free Ca²⁺ may act as a second messenger in plants much as it does in animal cells.     

Voltage-sensing phosphatase reveals temporal regulation of TRPC3/C6/C7 channels by membrane phosphoinositides

TRPC3/C6/C7 channels, a subgroup of classical/canonical TRP channels, are activated by diacylglycerol produced via activation of phospholipase C (PLC)-coupled receptors. Recognition of the physiological importance of these channels has been steadily growing, but the mechanism by which they are regulated remains largely unknown. We recently used a membrane-resident danio rerio voltage-sensing phosphatase (DrVSP) to study TRPC3/C6/C7 regulation and found that the channel activity was controlled by PtdIns(4,5)P 2-DAG signaling in a self-limiting manner (Imai Y et al., the Journal of Physiology, 2012). In this addendum, we present the advantages of using DrVSP as a molecular tool to study PtdIns(4,5)P 2 regulation. DrVSP should be readily applicable for studying phosphoinositide metabolism-linked channel regulation as well as lipid dynamics. Furthermore, in comparison to other modes of self-limiting ion channel regulation, the regulation of TRPC3/C6/C7 channels seems highly susceptible to activation signal strength, which could potentially affect both open duration and the time to peak activation and inactivation. Dysfunction of such self-limiting regulation may contribute to the pathology of the cardiovascular system, gastrointestinal tract and brain, as these channels are broadly distributed and affected by numerous neurohormonal agonists.     

Voltage-sensing phosphatase reveals temporal regulation of TRPC3/C6/C7 channels by membrane phosphoinositides

TRPC3/C6/C7 channels, a subgroup of classical/canonical TRP channels, are activated by diacylglycerol produced via activation of phospholipase C (PLC)-coupled receptors. Recognition of the physiological importance of these channels has been steadily growing, but the mechanism by which they are regulated remains largely unknown. We recently used a membrane-resident danio rerio voltage-sensing phosphatase (DrVSP) to study TRPC3/C6/C7 regulation and found that the channel activity was controlled by PtdIns(4,5)P₂-DAG signaling in a self-limiting manner (Imai Y et al., the Journal of Physiology, 2012). In this addendum, we present the advantages of using DrVSP as a molecular tool to study PtdIns(4,5)P₂ regulation. DrVSP should be readily applicable for studying phosphoinositide metabolism-linked channel regulation as well as lipid dynamics. Furthermore, in comparison to other modes of self-limiting ion channel regulation, the regulation of TRPC3/C6/C7 channels seems highly susceptible to activation signal strength, which could potentially affect both open duration and the time to peak activation and inactivation. Dysfunction of such self-limiting regulation may contribute to the pathology of the cardiovascular system, gastrointestinal tract and brain, as these channels are broadly distributed and affected by numerous neurohormonal agonists.     

TRPC3 and TRPC6 are essential for normal mechanotransduction in subsets of sensory neurons and cochlear hair cells

Transient receptor potential (TRP) channels TRPC3 and TRPC6 are expressed in both sensory neurons and cochlear hair cells. Deletion of TRPC3 or TRPC6 in mice caused no behavioural phenotype, although loss of TRPC3 caused a shift of rapidly adapting (RA) mechanosensitive currents to intermediate-adapting currents in dorsal root ganglion sensory neurons. Deletion of both TRPC3 and TRPC6 caused deficits in light touch and silenced half of small-diameter sensory neurons expressing mechanically activated RA currents. Double TRPC3/TRPC6 knock-out mice also showed hearing impairment, vestibular deficits and defective auditory brain stem responses to high-frequency sounds. Basal, but not apical, cochlear outer hair cells lost more than 75 per cent of their responses to mechanical stimulation. FM1-43-sensitive mechanically gated currents were induced when TRPC3 and TRPC6 were co-expressed in sensory neuron cell lines. TRPC3 and TRPC6 are thus required for the normal function of cells involved in touch and hearing, and are potential components of mechanotransducing complexes.