Identification of two domains involved in the assembly of transient receptor potential canonical channels

Transient receptor potential canonical (TRPC) channels are associated with calcium entry activity in nonexcitable cells. TRPCs can form homo- or heterotetrameric channels, in which case they can assemble together within a subfamily groups. TRPC1, 4, and 5 represent one group, and TRPC3, 6, and 7 represent the other. The molecular determinants involved in promoting subunit tetramerization are not known. To identify them, we generated chimeras by swapping the different domains of TRPC4 with the same regions in TRPC6. We showed that TRPC4 coimmunoprecipitated with the chimeras containing the ankyrin repeats and coiled-coil domains of TRPC4 into TRPC6. However, chimeras containing only the ankyrin repeats or only the coiled-coil domain of TRPC4 did not coimmunoprecipitate with TRPC4. We also showed that a second domain of interaction composed of the pore region and the C-terminal tail is involved in the oligomerization of TRPC4. However, chimeras containing only the pore region or only the C-terminal tail of TRPC4 did not coimmunoprecipitate with TRPC4. Furthermore, we showed that the N terminus of TRPC6 coimmunoprecipitated with the C terminus of TRPC6. Overexpression in HEK293T cells of chimeras that contained an N terminus and a C terminus from different subfamily groups increased intracellular calcium entry subsequent to stimulation of G(q) protein-coupled receptors. These results suggest that two types of interactions are involved in the assembly of the four subunits of the TRPC channel. The first interaction occurs between the N termini and involves two regions. The second interaction occurs between the N terminus and the C terminus and does not appear to be necessary for the activity of TRPCs.     

Multiple sites of interaction between the intracellular domains of an inwardly rectifying potassium channel, Kir6.2.

The amino-terminal and carboxy-terminal domains of inwardly rectifying potassium channel (Kir) subunits are both intracellular. A direct physical interaction between these two domains is involved in the response of Kir channels to regulatory factors such as G-proteins, nucleotides and intracellular pH. We have previously mapped the region within the N-terminal domain of Kir6.2 that interacts with the C-terminus. In this study we use a similar in vitro protein-protein interaction assay to map the regions within the C-terminus which interact with the N-terminus. We find that multiple interaction domains exist within the C-terminus: CID1 (amino acids (aa) 279-323), CID2 (aa 214-222) and CID3 (aa 170-204). These domains correlate with regions previously identified as making important contributions to Kir channel assembly and function. The highly conserved nature of the C-terminus suggests that a similar association with the N-terminus may be a feature common to all members of the Kir family of potassium channels, and that it may be involved in gating of Kir channels by intracellular ligands.     

D2 dopamine receptor homodimerization is mediated by multiple sites of interaction,including an intermolecular interaction involving transmembrane domain 4

In this study, we examined the mechanisms of intermolecular interaction involved in D2 dopamine receptor dimer formation to develop an understanding of the quaternary structure of G protein-coupled receptors. The potential role of two mechanisms was investigated: disulfide bridges and hydrophobic interactions between transmembrane domains. D2 dopamine receptor oligomers were unaffected by treatment with a reducing agent; however, oligomers of the D1 dopamine receptor dissociated following a similar treatment. This observation suggested that other forces such as hydrophobic interactions were more robust in the D2 receptor than in the D1 receptor in maintaining oligomerization. To elucidate which transmembrane domains were involved in the intermolecular hydrophobic interactions, truncation mutants were generated by successive deletion of transmembrane domains from amino and/or carboxyl portions of the D2 dopamine receptor. Immunoblot analyses revealed that all the fragments were well expressed but only fragments containing transmembrane domain 4 were able to self-associate, suggesting that critical areas for receptor dimerization resided within this transmembrane domain. Disruption of the helical structure of transmembrane domain 4 in a truncated receptor capable of forming dimers interfered with its ability to self-associate; however, a similar disruption of the transmembrane domain 4 helix structure in the full-length receptor did not significantly affect dimerization. These results indicated that there are other sites of interaction involved in D2 receptor oligomer assembly in addition to transmembrane domain 4.     

Formation of novel TRPC channels by complex subunit interactions in embryonic brain

Mammalian short TRP channels (TRPCs) are putative receptor- and store-operated cation channels that play a fundamental role in the regulation of cellular Ca2+ homeostasis. Assembly of the seven TRPC homologs (TRPC1-7) into homo- and heteromers can create a large variety of different channels. However, the compositions as well as the functional properties of native TRPC complexes are largely undefined. We performed a systematic biochemical study of TRPC interactions in mammalian brain and identified previously unrecognized channel heteromers composed of TRPC1, TRPC4, or TRPC5 and the diacylglycerol-activated TRPC3 or TRPC6 subunits. The novel TRPC heteromers were found exclusively in embryonic brain. In heterologous systems, we demonstrated that assembly of these novel heteromers required the combination of TRPC1 plus TRPC4 or TRPC5 subunits along with diacylglycerol-sensitive subunits in the channel complexes. Functional interaction of the TRPC subunits was verified using a dominant negative TRPC5 mutant (TRPC5DN). Co-expression of TRPC5DN suppressed currents through TRPC5- and TRPC4-containing complexes; TRPC3-associated currents were unaffected by TRPC5DN unless TRPC1 was also co-expressed. This complex assembly mechanism increases the diversity of TRPC channels in mammalian brain and may generate novel heteromers that have specific roles in the developing brain.     

The dynamic complexity of the TRPC1 channelosome

A rise in cytoplasmic [Ca2+] due to store-operated Ca2+ entry (SOCE) triggers a plethora of responses, both acute and long term. This leads to the important question of how this initial signal is decoded to regulate specific cellular functions. It is now clearly established that local [Ca2+] at the site of SOCE can vary significantly from the global [Ca2+] in the cytosol. Such Ca2+ microdomains are generated by the assembly of key Ca2+ signaling proteins within the domains. For example, GPCR, IP 3 receptors, TRPC3 channels, the plasma membrane Ca2+ pump and the endoplasmic reticulum (ER) Ca2+ pump have all been found to be assembled in a complex and all of them contribute to the Ca2+ signal. Recent studies have revealed that two other critical components of SOCE, STIM1 and Orai1, are also recruited to these regions. Thus, the entire machinery for activation and regulation of SOCE is compartmentalized in specific cellular domains which facilitates the specificity and rate of protein-protein interactions that are required for activation of the channels. In the case of TRPC1-SOC channels, it appears that specific lipid domains, lipid raft domains (LRDs), in the plasma membrane, as well as cholesterol-binding scaffolding proteins such as caveolin-1 (Cav-1), are involved in assembly of the TRPC channel complexes. Thus, plasma membrane proteins and lipid domains as well as ER proteins contribute to the SOCE-Ca2+ signaling microdomain and modulation of the Ca2+ signals per se. Of further interest is that modulation of Ca2+ signals, i.e. amplitude and/or frequency, can result in regulation of specific cellular functions. The emerging data reveal a dynamic Ca2+ signaling complex composed of TRPC1/Orai1/STIM1 that is physiologically consistent with the dynamic nature of the Ca2+ signal that is generated. This review will focus on the recent studies which demonstrate critical aspects of the TRPC1 channelosome that are involved in the regulation of TRPC1 function and TRPC1-SOC-generated Ca2+ signals.     

Junctate, an inositol 1,4,5-triphosphate receptor associated protein, is present in rodent sperm and binds TRPC2 and TRPC5 but not TRPC1 channels

The acrosome reaction, the first step of the fertilization, is induced by calcium influx through Canonical Transient Receptor Potential channels (TRPC). The molecular nature of TRPC involved is still a debated question. In mouse, TRPC2 plays the most important role and is responsible for the calcium plateau. However, TRPC1 and TRPC5 are also localized in the acrosomal crescent of the sperm head and may participate in calcium signaling, especially in TRPC2-deficient mice. Activation of TRPC channels is an unresolved question in germ and somatic cells as well. In particular, in sperm, little is known concerning the molecular events leading to TRPC2 activation. From the discovery of IP3R binding domains on TRPC2, it has been suggested that TRPC channel activation may be due to a conformational coupling between IP3R and TRPC channels. Moreover, recent data demonstrate that junctate, an IP3R associated protein, participates also in the gating of some TRPC. In this study, we demonstrate that junctate is expressed in sperm and co-localizes with the IP3R in the acrosomal crescent of the anterior head of rodent sperm. Consistent with its specific localization, we show by pull-down experiments that junctate interacts with TRPC2 and TRPC5 but not with TRPC1. We focused on the interaction between TRPC2 and junctate, and we show that the N-terminus of junctate interacts with the C-terminus of TRPC2, both in vitro and in a heterologous expression system. We show that junctate binds to TRPC2 independently of the calcium concentration and that the junctate binding site does not overlap with the common IP3R/calmodulin binding sites. TRPC2 gating is downstream phospholipase C activation, which is a key and necessary step during the acrosome reaction. TRPC2 may then be activated directly by diacylglycerol (DAG), as in neurons of the vomeronasal organ. In the present study, we investigated whether DAG could promote the acrosome reaction. We found that 100 microM OAG, a permeant DAG analogue, was unable to trigger the acrosome reaction. Altogether, these results provide a new hypothesis concerning sperm TRPC2 gating: TRPC2 activation may be due to modifications of its interaction with both junctate and IP3R, induced by depletion of calcium from the acrosomal vesicle.     

Identification of a Membrane-targeting Domain of the Transient Receptor Potential Canonical (TRPC)4 Channel Unrelated to Its Formation of a Tetrameric Structure

Canonical transient receptor potential (TRPC) channels are Ca²⁺-permeable nonselective cation channels that are activated by a wide variety of stimuli, including G protein-coupled receptors (GPCRs). The TRPC4 channel is expressed in a punctate distribution in the membrane. To identify the regulating region of the channel trafficking to the membrane, we generated deletion mutants of the TRPC4 channel. We determined that when either region that was downstream of the 20 amino acids of the N terminus or the 700–730 amino acids was deleted, the mutants were retained in the endoplasmic reticulum. By coexpression of the wild-type TRPC4 with deletion mutants, we found that the 23–29 amino acids of the N terminus regulate a membrane trafficking. Additionally, by the fluorescence resonance energy transfer (FRET) method, we found that the regions downstream of the 99 amino acid region of the N terminus and upstream of the 730 amino acid region in the C terminus produce assembly of the TRPC4 tetramers. We inferred the candidate proteins that regulate or interact with the 23–29 domain of TRPC4.     

Enhanced Trafficking of Tetrameric Kv4.3 Channels by KChIP1 Clamping

The cytoplamsic auxiliary KChIPs modulate surface expression and gating properties of Kv4 channels. Recent co-crystal structure of Kv4.3 N-terminus and KChIP1 reveals a clamping action of the complex in which a single KChIP1 molecule laterally binds two neighboring Kv4.3 N-termini at different locations, thus forming two contact interfaces involved in the protein–protein interaction. In the second interface, it functions to stabilize the tetrameric assembly, but the role it plays in channel trafficking remains elusive. In this study, we examined the effects of KChIP1 on Kv4 protein trafficking in COS-7 cells expressing EGFP-tagged Kv4.3 channels using confocal microscopy. Mutations either in KChIP1 (KChIP1 L39E-Y57A-K61A) or Kv4.3 (Kv4.3 E70A-F73E) that disrupt the protein–protein interaction within the second interface can reduce surface expression of Kv4 channel proteins. Kv4.3 C110A, the Zn²⁺ binding site mutation in T1 domain, that disrupts the tetrameric assembly of the channels can be rescued by WT KChIP1, but not the KChIP1 triple mutant. These results were further confirmed by whole cell current recordings in oocytes. Our findings show that key residues of second interface involved in stabilizing tetrameric assembly can regulate the channel trafficking, indicating an intrinsic link between tetrameric assembly and channel trafficking. The results also suggest that formation of octameric Kv4 and KChIP complex by KChIPs clamping takes place before their trafficking to final destination on the cell surface. Special issue article in honor of Dr. Ji-Sheng Han.     

Dimerization of TWIK-1 K+ channel subunits via a disulfide bridge.

TWIK-1 is a new type of K+ channel with two P domains and is abundantly expressed in human heart and brain. Here we show that TWIK-1 subunits can self-associate to give dimers containing an interchain disulfide bridge. This assembly involves a 34 amino acid domain that is localized to the extracellular M1P1 linker loop. Cysteine 69 which is part of this interacting domain is implicated in the formation of the disulfide bond. Replacing this cysteine with a serine residue results in the loss of functional K+ channel expression. This is the first example of a covalent association of functional subunits in voltage-sensitive channels via a disulfide bridge.     

Canonical Transient Receptor Potential (TRPC) 1 Acts as a Negative Regulator for Vanilloid TRPV6-mediated Ca2+ Influx

TRP proteins mostly assemble to homomeric channels but can also heteromerize, preferentially within their subfamilies. The TRPC1 protein is the most versatile member and forms various TRPC channel combinations but also unique channels with the distantly related TRPP2 and TRPV4. We show here a novel cross-family interaction between TRPC1 and TRPV6, a Ca²⁺ selective member of the vanilloid TRP subfamily. TRPV6 exhibited substantial co-localization and in vivo interaction with TRPC1 in HEK293 cells, however, no interaction was observed with TRPC3, TRPC4, or TRPC5. Ca²⁺ and Na⁺ currents of TRPV6-overexpressing HEK293 cells are significantly reduced by co-expression of TRPC1, correlating with a dramatically suppressed plasma membrane targeting of TRPV6. In line with their intracellular retention, remaining currents of TRPC1 and TRPV6 co-expression resemble in current-voltage relationship that of TRPV6. Studying the N-terminal ankyrin like repeat domain, structurally similar in the two proteins, we have found that these cytosolic segments were sufficient to mediate a direct heteromeric interaction. Moreover, the inhibitory role of TRPC1 on TRPV6 influx was also maintained by expression of only its N-terminal ankyrin-like repeat domain. Our experiments provide evidence for a functional interaction of TRPC1 with TRPV6 that negatively regulates Ca²⁺ influx in HEK293 cells.     

The dynamic complexity of the TRPC1 channelosome

A rise in cytoplasmic [Ca²⁺] due to store-operated Ca²⁺ entry (SOCE) triggers a plethora of responses, both acute and long term. This leads to the important question of how this initial signal is decoded to regulate specific cellular functions. It is now clearly established that local [Ca²⁺] at the site of SOCE can vary significantly from the global [Ca²⁺] in the cytosol. Such Ca²⁺ microdomains are generated by the assembly of key Ca²⁺ signaling proteins within the domains. For example, GPCR, IP₃ receptors, TRPC3 channels, the plasma membrane Ca²⁺ pump and the endoplasmic reticulum (ER) Ca²⁺ pump have all been found to be assembled in a complex and all of them contribute to the Ca²⁺ signal. Recent studies have revealed that two other critical components of SOCE, STIM1 and Orai1, are also recruited to these regions. Thus, the entire machinery for activation and regulation of SOCE is compartmentalized in specific cellular domains which facilitates the specificity and rate of protein-protein interactions that are required for activation of the channels. In the case of TRPC1-SOC channels, it appears that specific lipid domains, lipid raft domains (LRDs), in the plasma membrane, as well as cholesterol-binding scaffolding proteins such as caveolin-1 (Cav-1), are involved in assembly of the TRPC channel complexes. Thus, plasma membrane proteins and lipid domains as well as ER proteins contribute to the SOCE-Ca²⁺ signaling microdomain and modulation of the Ca²⁺ signals per se. Of further interest is that modulation of Ca²⁺ signals, i.e. amplitude and/or frequency, can result in regulation of specific cellular functions. The emerging data reveal a dynamic Ca²⁺ signaling complex composed of TRPC1/Orai1/STIM1 that is physiologically consistent with the dynamic nature of the Ca²⁺ signal that is generated. This review will focus on the recent studies which demonstrate critical aspects of the TRPC1 channelosome that are involved in the regulation of TRPC1 function and TRPC1-SOC-generated Ca²⁺ signals.     

MxA, a member of the dynamin superfamily, interacts with the ankyrin-like repeat domain of TRPC

Mammalian transient receptor potential canonical channels have been proposed as the molecular entities associated with calcium entry activity in nonexcitable cells. Amino acid sequence analyses of TRPCs revealed the presence of ankyrin-like repeat domains, one of the most common protein-protein interaction motifs. Using a yeast two-hybrid interaction assay, we found that the second ankyrin-like repeat domain of TRPC6 interacted with MxA, a member of the dynamin superfamily. Using a GST pull-down and co-immunoprecipitation assay, we showed that MxA interacted with TRPC1, -3, -4, -5, -6, and -7. Overexpression of MxA in HEK293T cells slightly increased endogenous calcium entry subsequent to stimulation of G(q) protein-coupled receptors or store depletion by thapsigargin. Co-expression of MxA with TRPC6 enhanced agonist-induced or OAG-induced calcium entry activity. GTP binding-defective MxA mutants had only a minor potentiating effect on OAG-induced TRPC6 activity. However, a MxA mutant that could bind GTP but that lacked GTPase activity produced the same effect as MxA on OAG-induced TRPC6 activity. These results indicated that MxA interacted specifically with the second ankyrin-like repeat domain of TRPCs and suggested that monomeric MxA regulated the activity of TRPC6 by a mechanism requiring GTP binding. Additional results showed that an increase in the endogenous expression of MxA, induced by a treatment with interferon alpha, regulated the activity of TRPC6. The study clearly identified MxA as a new regulatory protein involved in Ca2+ signaling.     

Regulation of TRPC6 channels by non-steroidal anti-inflammatory drugs

Family focal segmental glomerulosclerosis (FSGS) is characterized by sclerosis and hyalinosis of particular loops of glomeruli and is one of the causes of the nephrotic syndrome. Certain mutations in the structure of TRPC6 channels are the genetic impetus for FSGS development resulting in podocytes functional abnormalities and various nephropathies. We have recently demonstrated that non-steroid antiinflammatory drugs (NSAID) ibuprofen and diclofenac decrease the activity of endogenous TRPC-like calcium channels in the podocytes of the freshly isolated rat glomeruli. It has also been shown that TRPC6 channels are expressed in the podocytes. In the current study we have functionally reconstituted TRPC6 channels in mammalian cells to investigate the effects of diclofenac on the activity of wild type TRPC6 channel and TRPC6^P112Q channel containing a mutation in the N-terminus that was described in FSGS patients. Intracellular calcium level measurements in transfected cells revealed a more intensive carbachol-induced increase of calcium concentration in HEK-293 cells expressing TRPC6^P112Q versus the cells expressing wild-type TRPC6. We also performed patch-clamp experiments to study TRPC6 channels reconstituted in Chinese hamster ovary (CHO) cell line and found that application of diclofenac (500 μM) acutely reduced single channel activity. Preincubation with diclofenac (100 μM) also decreased the whole-cell current in CHO cells overexpressing TRPC6^P112Q. Therefore, our previously published data on the effects of NSAID on TRPC-like channels in the isolated rat glomeruli, along with this current investigation on the cultured overexpressed mammalian cells, allows hypothesizing that TRPC6 channels may be a target for NSAID that can be important in the treatment of FSGS.     

Recent progress in structural studies on canonical TRP ion channels

Canonical TRP channels (TRPC) are non-selective cation channels that are involved in various important physiological processes. Currently, the structures of different TRPC ion channel family members are resolved by cryo-EM at resolutions ranging from 2.8 Å to 5.8 Å. These structures reveal the conserved architecture of TRPC ion channels as well as the specific features of each channel subtype. This review focuses on the structural differences in the extracellular portions, transmembrane domains and the cytoplasmic domains of TRPC channels.     

Analysis of TRPC3-interacting proteins by tandem mass spectrometry

Mammalian transient receptor potential canonical (TRPC) channels are a family of nonspecific cation channels that are activated in response to stimulation of phospholipase C (PLC)-dependent hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate. Despite extensive studies, the mechanism(s) involved in regulation of mammalian TRPC channels remains unknown. Presence of various protein-interacting domains in TRPC channels have led to the suggestion that they associate with proteins that are involved in their function and regulation. This study was directed toward identifying the proteins associated with native TRPC3 using a shotgun proteomic approach. Anti-TRPC3 antibody was used to immunoprecipitate TRPC3 from solubilized rat brain crude membranes under conditions that allow retention of TRPC3 function. Proteins in the TRPC3 (using anti-TRPC3 antibody) and control (using rabbit IgG) immunoprecipitates were separated by SDS-PAGE, the gel was sectioned, and the resolved proteins were digested by trypsin in situ. After extraction of the peptides, the peptides were separated by HPLC and sequences derived by MS/MS. Analysis of the data revealed 64 specific TRPC3-associated proteins which can be grouped in terms of their cellular location and involvement in specific cellular function. Many of the proteins identified have been previously reported as TRPC3-regulatory proteins, such as IP3Rs and vesicle trafficking proteins. In addition, we report novel putative TRPC3-interacting proteins, including those involved in protein endocytosis and neuronal growth. To our knowledge, this is the first comprehensive proteomic analysis of a native TRPC channel. These data reveal potential TRPC3 regulatory proteins and provide novel insights of the mechanism(s) regulating TRPC3 channels as well as the possible cellular functions where the channel might be involved.     

Proteins modulating TRP channel function

TRP channels are involved in different signaling cascades; TRP channels can be activated via hormones and neurotransmitter in a receptor/G-protein-mediated manner or by osmotic, thermic or mechanic stimuli. The overall functional role of TRP channels within these processes of hormonal cellular control, nociception or cellular calcium homeostasis is still unclear, as these complex processes often involve macromolecular structures. Whereas the integration of Drosophila TRP in the phototransduction process is becoming clear, the understanding of the participation of mammalian TRP channels in signal transduction complexes is only beginning. TRP channels have been demonstrated to interact with PDZ domain proteins, and both scaffold and regulatory function have been shown for INAD, the PDZ domain protein of the Drosophila phototransduction complex. In mammalian cells, the interaction of NHERF and TRPC4 has been shown and it is anticipated that NHERF may abolish the apparent store-dependent regulation of TRPC4 and TRPC5. Whereas TRP channels and PDZ domain proteins form permanent heterodimeric proteins, the interaction of calcium-binding proteins is dependent on the calcium concentration and is, therefore, dynamic. The prototype of calcium-binding protein used for experiments is calmodulin; whether or not calmodulin is also the natural interaction partner of TRP channels is an open question.     

Conserved Gating Elements in TRPC4 and TRPC5 Channels

TRPC4 and TRPC5 proteins share 65% amino acid sequence identity and form Ca²⁺-permeable nonselective cation channels. They are activated by stimulation of receptors coupled to the phosphoinositide signaling cascade. Replacing a conserved glycine residue within the cytosolic S4–S5 linker of both proteins by a serine residue forces the channels into an open conformation. Expression of the TRPC4_G503S and TRPC5_G504S mutants causes cell death, which could be prevented by buffering the Ca²⁺ of the culture medium. Current-voltage relationships of the TRPC4_G503S and TRPC5_G504S mutant ion channels resemble that of fully activated TRPC4 and TRPC5 wild-type channels, respectively. Modeling the structure of the transmembrane domains and the pore region (S4-S6) of TRPC4 predicts a conserved serine residue within the C-terminal sequence of the predicted S6 helix as a potential interaction site. Introduction of a second mutation (S623A) into TRPC4_G503S suppressed the constitutive activation and partially rescued its function. These results indicate that the S4–S5 linker is a critical constituent of TRPC4/C5 channel gating and that disturbance of its sequence allows channel opening independent of any sensor domain.     

Selective Gαi subunits as novel direct activators of transient receptor potential canonical (TRPC)4 and TRPC5 channels

The ubiquitous transient receptor potential canonical (TRPC) channels function as non-selective, Ca(2+)-permeable channels and mediate numerous cellular functions. It is commonly assumed that TRPC channels are activated by stimulation of Gα(q)-PLC-coupled receptors. However, whether the Gα(q)-PLC pathway is the main regulator of TRPC4/5 channels and how other Gα proteins may regulate these channels are poorly understood. We previously reported that TRPC4/TRPC5 can be activated by Gα(i). In the current work, we found that Gα(i) subunits, rather than Gα(q), are the primary and direct activators of TRPC4 and TRPC5. We report a novel molecular mechanism in which TRPC4 is activated by several Gα(i) subunits, most prominently by Gα(i2), and TRPC5 is activated primarily by Gα(i3). Activation of Gα(i) by the muscarinic M2 receptors or expression of the constitutively active Gα(i) mutants equally and fully activates the channels. Moreover, both TRPC4 and TRPC5 are activated by direct interaction of their conserved C-terminal SESTD (SEC14-like and spectrin-type domains) with the Gα(i) subunits. Two amino acids (lysine 715 and arginine 716) of the TRPC4 C terminus were identified by structural modeling as mediating the interaction with Gα(i2). These findings indicate an essential role of Gα(i) proteins as novel activators for TRPC4/5 and reveal the molecular mechanism by which G-proteins activate the channels.     

Interaction between TRPC channel subunits in endothelial cells

Transient Receptor Potential Canonical (TRPC) proteins have been identified in mammals as a family of plasma membrane calcium-permeable channels activated by different kinds of stimuli in several cell types. We have studied TRPC subunit expression in bovine aortic endothelial (BAE-1) cells, where stimulation with basic fibroblast growth factor (bFGF), a potent angiogenetic factor, induces calcium entry carried at least partially by TRPC1 channels. By means of a RT-PCR approach, we have found that, in addition to TRPC1, only TRPC4 is expressed, both at the mRNA and protein level, as confirmed by immunoblotting and immunocytochemical analysis. Because functional TRPC channels are formed by assembly of four subunits in either homo- or heterotetrameric structures, we have carried out immunoprecipitation experiments and showed that TRPC1 and TRPC4 interact to form heteromers in these cells, independently from culture conditions (high or low percent of fetal calf serum, stimulation with bFGF). Moreover, the data show that TRPC subunits are not tyrosine-phosphorylated after bFGF stimulation and they do not co-immunoprecipitate with the type 1 FGF receptor. These results suggest that BAE-1 cells are a suitable model to study function and regulation of endogenous TRPC1/TRPC4 heteromers.