Expression of TRPC4 channel protein that interacts with NHERF-2 in rat descending vasa recta

The PDZ domain adaptor protein Na⁺/H⁺ exchanger regulatory factor (NHERF)-2 is expressed in renal medullary descending vasa recta (DVR), although its function has not been defined. Transient receptor potential channels (TRPC) TRPC4 and TRPC5, nonselective cation channels that transport Ca²⁺, were recently demonstrated to complex with the NHERF proteins. We investigated whether TRPC4 and/or TRPC5 are associated with NHERF-2 in DVR. RT-PCR revealed mRNA for TRPC4 and NHERF-2, but not for TRPC5 or NHERF-1, in microdissected DVR. Immunohistochemical studies demonstrated expression of TRPC4 and NHERF-2 proteins in both the endothelial cells and pericytes. These proteins colocalized in some cells of the DVR. TRPC4 coimmunoprecipitated with NHERF-2 from renal medullary lysates, and NHERF-2 coimmunoprecipitated with TRPC4. TRPC5 was not detected in DVR with the use of immunohistochemistry or in NHERF-2 immunoprecipitates. We conclude that DVR pericytes and endothelia coexpress TRPC4 and NHERF-2 mRNA and protein and that these proteins colocalize and coimmunoprecipitate, indicating a possible physical association. These findings suggest that TRPC4 and NHERF-2 may play a role in interactions related to Ca²⁺ signaling. PDZ proteins; calcium channels; medulla; pericytes; endothelium; microcirculation     

Regulation of Drosophila TRPC channels by protein and lipid interactions

The Drosophila TRPC channels TRP and TRPL are the founding members of the TRP superfamily of ion channels, proteins likely to be important components of calcium influx pathways. The activation of these channels in the context of fly phototransduction is one of the few in vivo models for TRPC channel activation and has served as a paradigm for understanding TRPC function. TRP and TRPL are activated by G-protein coupled PI(4,5)P(2) hydrolysis through a mechanism in which IP(3) receptor mediated calcium release seems dispensable. Recent analysis has provided compelling evidence that the accurate turnover of PI(4,5)P(2) generated lipid messengers in essential for regulating TRP and TRPL activity. TRP channels also appear to exist in the context of a macromolecular complex containing key components involved in activation such as phospholipase Cbeta and protein kinase C. This complex may be important for activation. The role of these protein and lipid elements in regulating TRP and TRPL activity is discussed in this review.     

Calcium store contents control the expression of TRPC1, TRPC3 and TRPV6 proteins in LNCaP prostate cancer cell line

The mammalian homologues of the Drosophila transient receptor potential (TRP) represent a superfamily of ion channels involved in Ca(2+) homeostasis. Several members of this family are activated either by a depletion of the internal stores of Ca(2+) or by stimulation of G protein-coupled receptors. In androgen responsive prostate cancer cell line LNCaP, TRPC1, TRPC4 and/or TRPV6 have been reported to function as store-operated channels (SOCs) while TRPC3 might be involved in the response to agonist stimulation, possibly through the induction of diacylglycerol production by phospholipase C. However, the control of expression of these TRP proteins is largely unknown. In the present study, we have investigated if the expression of the TRP proteins possibly involved in the capacitative influx of calcium is influenced by the contents of Ca(2+) in the endoplasmic reticulum. Using real-time PCR and Western blot techniques, we show that the expression of TRPC1, TRPC3 and TRPV6 proteins increases after a prolonged (24-48 h) depletion of the stores with thapsigargin. The upregulation of TRPC1 and TRPC3 depends on the store contents level and involves the activation of the Ca(2+)/calmodulin/calcineurin/NFAT pathway. Functionally, cells overexpressing TRPC1, TRPC3 and TRPV6 channels after a prolonged depletion of the stores showed an increased [Ca(2+)](i) response to alpha-adrenergic stimulation. However, the store-operated entry of calcium was unchanged. The isolated overexpression of TRPV6 (without overexpression of TRPC1 and TRPC3) did not produce this increased response to agonists, therefore suggesting that TRPC1 and/or TRPC3 proteins are responsible for the response to alpha-adrenergic stimulation but that TRPC1, TPRC3 and TRPV6 proteins, expressed alone or concomitantly, are not sufficient for SOC formation.     

The TRPC3/6/7 subfamily of cation channels

The mammalian transient receptor potential (TRP) proteins consist of a superfamily of Ca2+-permeant non-selective cation channels with structural similarities to Drosophila TRP. The TRP superfamily can be divided into three major families, among them the "canonical TRP" family (TRPC). The seven protein products of the mammalian TRPC family of genes (designated TRPC1-7) share in common the activation through PLC-coupled receptors and have been proposed to encode components of native store-operated channels in different cell types. In addition, the three members of the TRPC3/6/7 subfamily of TRPC channels can be activated by diacylglycerol analogs, providing a possible mechanism of activation of these channels by PLC-coupled receptors. This review summarizes the current knowledge about the mechanism of activation of the TRPC3/6/7 subfamily, as well as the potential role of these proteins as components of native Ca2+-permeant channels.     

The roles of PDZ-containing proteins in PLC-beta-mediated signaling

Mammalian phospholipase C-beta isozymes are activated by a heterotrimeric GTP-binding protein linked to various cell surface receptors. Recent reports suggest that PDZ domain proteins play a significant role of PDZ-containing proteins in the regulation of mammalian PLC-beta isozymes. PDZ-containing proteins mediate the clustering of receptors and signaling molecules and thereby regulate agonist-induced signal transduction in polarized cells such as neuronal and epithelial cells. NORPA, a Drosophila PLC-beta, is known to be a component of a signaling complex that includes TRP and rhodopsin through interaction with INAD, a PDZ-containing protein. Mammalian PLC-beta1 and -beta2 isoforms interact with a PDZ-containing protein NHERF which is coupled to Trp4, a Ca(2+) channel. In addition, PLC-beta3 specifically interacts with E3KARP, another protein closely related to NHERF, through its C-terminal PDZ-binding motif. E3KARP up-regulates the PLC-beta3 activation coupled to muscarinic receptor. In this review, the role of signaling complexes mediated by PDZ-containing proteins in the regulation of PLC-beta isoforms will be discussed.     

Regulation of Drosophila TRPC channels by protein and lipid interactions

The Drosophila TRPC channels TRP and TRPL are the founding members of the TRP superfamily of ion channels, proteins likely to be important components of calcium influx pathways. The activation of these channels in the context of fly phototransduction is one of the few in vivo models for TRPC channel activation and has served as a paradigm for understanding TRPC function. TRP and TRPL are activated by G-protein coupled PI(4,5)P₂ hydrolysis through a mechanism in which IP₃ receptor mediated calcium release seems dispensable. Recent analysis has provided compelling evidence that the accurate turnover of PI(4,5)P₂ generated lipid messengers in essential for regulating TRP and TRPL activity. TRP channels also appear to exist in the context of a macromolecular complex containing key components involved in activation such as phospholipase Cβ and protein kinase C. This complex may be important for activation. The role of these protein and lipid elements in regulating TRP and TRPL activity is discussed in this review.     

Association of mammalian trp4 and phospholipase C isozymes with a PDZ domain-containing protein, NHERF

Mammalian homologues of Drosophila Trp have been implicated to form channels that are activated following the depletion of Ca(2+) from internal stores. Recent studies indicate that actin redistribution is required for the activation of these channels. Here we show that murine Trp4 and Trp5, as well as phospholipase C beta1 and beta2 interact with the first PDZ domain of NHERF, regulatory factor of the Na(+)/H(+) exchanger. We demonstrated the association of Trp4 and phospholipase C-beta1 with NHERF in vivo in an HEK293 cell line expressing Trp4 and in adult mouse brain by immuno-coprecipitation. NHERF is a two PDZ domain-containing protein that associates with the actin cytoskeleton via interactions with members of ezrin/radixin/moesin family. Thus, store-operated channels involving Trp4 and Trp5 can form signaling complexes with phospholipase C isozymes via interactions with NHERF and thereby linking the lipase and the channels to the actin cytoskeleton. The interaction with the PDZ protein may constitute an important mechanism for distribution and regulation of store-operated channels.     

Assembly and trafficking of a multiprotein ROMK (Kir 1.1) channel complex by PDZ interactions

The ROMK subtypes of inward rectifier K+ channels (Kir 1.1, KCNJ1) mediate potassium secretion and regulate NaCl reabsorption in the kidney. In the present study, the role of the PDZ binding motif in ROMK function is explored. Here we identify the Na/H exchange regulatory factors, NHERF-1 and NHERF-2, as PDZ domain interaction partners of the ROMK channel. Characterization of the basis and consequences of NHERF association with ROMK reveals a PDZ interaction-dependent trafficking process and a coupling mechanism for linking ROMK to a channel modifier protein, the cystic fibrosis transmembrane regulator (CFTR). As measured by antibody binding of external epitope-tagged forms of Kir 1.1 in intact cells, NHERF-1 or NHERF-2 coexpression increased cell surface expression of ROMK. Channel interaction with NHERF proteins and effects of NHERF on ROMK localization were dependent on the presence of the PDZ domain binding motif in ROMK. Both NHERF proteins contain two PDZ domains; recombinant protein-protein binding assays and yeast-two-hybrid studies revealed that ROMK preferentially associates with the second PDZ domain of NHERF-1 and with the first PDZ domain of NHERF-2, precisely opposite of what has been reported for CFTR. Consistent with the scaffolding capacity of the NHERF proteins, coexpression of NHERF-2 with ROMK and CFTR dramatically increases the amount of ROMK protein that coimmunopurifies and functionally interacts with CFTR. Thus NHERF facilitates assembly of a ternary complex containing ROMK and CFTR. These observations raise the possibility that PDZ-based interactions may underscore physiological regulation and membrane targeting of ROMK in the kidney.     

Cytoskeletal reorganization internalizes multiple transient receptor potential channels and blocks calcium entry into human neutrophils

Store-operated calcium entry (SOCE) is required for polymorphonuclear neutrophil (PMN) activation in response to G protein-coupled agonists. Some immunocytes express proteins homologous to the Drosophila transient receptor potential gene (trp) calcium channel. TRP proteins assemble into heterotetrameric ion channels and are known to support SOCE in overexpression systems, but the evidence that TRP proteins support SOCE and are functionally important in wild-type cells remains indirect. We therefore studied the expression and function of TRP proteins in primary human PMN. TRPC1, TRPC3, TRPC4, and TRPC6 were all expressed as mRNA as well as membrane proteins. Immunofluorescence microscopy demonstrated localization of TRPC1, TRPC3, and TRPC4 to the PMN cell membrane and their internalization after cytoskeletal reorganization by calyculin A (CalyA). Either TRPC internalization by CalyA or treatment with the inositol triphosphate receptor inhibitor 2-aminoethoxydiphenyl borane resulted in the loss of PMN SOCE. Cytochalasin D (CytoD) disrupts actin filaments, thus preventing cytoskeletal reorganization, and pretreatment with CytoD rescued PMN SOCE from inhibition by CalyA. Comparative studies of CytoD and 2-aminoethoxydiphenyl borane inhibition of PMN cationic entry after thapsigargin or platelet-activating factor suggested that SOCE occurs through both calcium-specific and nonspecific pathways. Taken together, these studies suggest that the multiple TRPC proteins expressed by human PMN participate in the formation of at least two store-operated calcium channels that have differing ionic permeabilities and regulatory characteristics.     

Enkurin is a novel calmodulin and TRPC channel binding protein in sperm

The TRPC cation channel family has been implicated in receptor- or phospholipase C (PLC)-mediated Ca2+ entry into animal cells. These channels are present in mammalian sperm and are assigned a role in ZP3-evoked Ca2+ influx that drives acrosome reactions. However, the mechanisms controlling channel activity and coupling Ca2+ entry through these channels to cellular responses are not well understood. A yeast two-hybrid screen was carried out to identify TRPC-interacting proteins that would be candidate regulators or effectors. We identified a novel protein, enkurin, that is expressed at high levels in the testis and vomeronasal organ and at lower levels in selected other tissues. Enkurin interacts with several TRPC proteins (TRPC1, TRPC2, TRPC5, but not TRPC3) and colocalizes with these channels in sperm. Three protein-protein interaction domains were identified in enkurin: a C-terminal region is essential for channel interaction; an IQ motif binds the Ca2+ sensor, calmodulin, in a Ca2+-dependent manner; and a proline-rich N-terminal region contains predicted ligand sequences for SH3 domain proteins, including the SH3 domain of the p85 regulatory subunit of 1-phosphatidylinositol-3-kinase. We suggest that enkurin is an adaptor that functions to localize a Ca2+ sensitive signal transduction machinery in sperm to a Ca2+-permeable ion channel.     

The mammalian TRPC cation channels

Transient Receptor Potential-Canonical (TRPC) channels are mammalian homologs of Transient Receptor Potential (TRP), a Ca(2+)-permeable channel involved in the phospholipase C-regulated photoreceptor activation mechanism in Drosophila. The seven mammalian TRPCs constitute a family of channels which have been proposed to function as store-operated as well as second messenger-operated channels in a variety of cell types. TRPC channels, together with other more distantly related channel families, make up the larger TRP channel superfamily. This review summarizes recent findings on the structure, regulation and function of the apparently ubiquitous TRPC cation channels.     

Regulation of Drosophila TRPC channels by lipid messengers

The Drosophila TRPC channels TRP and TRPL are the founding members of the TRP superfamily of ion channels, which are important components of calcium influx pathways in virtually all cells. The activation of these channels in the context of fly phototransduction is one of the few in vivo models for TRPC channel activation and has served as a paradigm for understanding TRPC function. TRP and TRPL are activated by G-protein coupled PIP₂ hydrolysis through a mechanism in which IP₃ receptor mediated calcium release seems dispensable. Recent analysis has provided compelling evidence that one or more PIP₂ generated lipid messengers, as well as PIP₂ itself, are essential for regulating TRP and TRPL activity. Evidence on the role of these lipid elements in regulating TRP and TRPL activity is discussed in this review.     

Association of immunophilins with mammalian TRPC channels

Drosophila photoreceptor channels TRP and TRPL are held in a large signalplex by the scaffolding protein, INAD. Immunophilin FKBP59, another member of the signalplex, binds to both INAD and TRPL. Mutation P702Q or P709Q in the highly conserved TRPL sequence (701)LPPPFNVLP(709), eliminates TRPL interaction with FKBP59. The first leucylprolyl (LP) dipeptide in this region is conserved in mammalian TRPC channel proteins. However, the second LP is changed to isoleucylprolyl (IP) in TRPC1, -C4, and -C5, and valylprolyl (VP) in TRPC3, -C6, and -C7. The purpose of the present study was to determine if mammalian FKBP12 or FKBP52 interact with TRPC channel proteins. Using TRPC-specific antibodies, immunoprecipitations from Sf9 cells individually co-expressing each of the TRPC proteins along with the immunophilins showed that TRPC3, -C6, and -C7 interact with FKBP12, whereas TRPC1, -C4, and -C5 interact with FKBP52. The binding of FKBP12 and FKBP52 was specific and could be displaced by the immunosuppressant drug FK506, at concentrations of 0.5 and 10 microm, respectively. To evaluate TRPC-immunophilin interactions in vivo, immunoprecipitations were performed using membrane lysates of rat cerebral cortex. FKBP12 co-immunoprecipitated with TRPC3, -C6, and -C7 from rat brain, whereas FKBP52 was found to associate with TRPC1, -C4, and -C5. The association of immunophilins with the TRPC channels in rat brain lysates could be displaced by FK506. Receptor-mediated activation of TRPC6, stably expressed in HEK cells, was significantly inhibited by FK506, which also disrupted interaction between TRPC6 and the endogenous immunophilin found in HEK cells. Pro to Gln mutations in the first LP dipeptide in the putative FKBP binding domain eliminated FKBP12 and FKBP52 interaction with TRPC3 and -C6, and TRPC1 and -C4, respectively. However, mutual swap of VP and IP in TRPC3 and TRPC5 did not alter the association or the selectivity of the channels for their respective immunophilin binding partner. These results suggest that immunophilins are TRPC channel accessory proteins that play an important role in the mechanism of channel activation following receptor stimulation.     

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.     

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.     

Crystal structure of the PDZ1 domain of human Na(+)/H(+) exchanger regulatory factor provides insights into the mechanism of carboxyl-terminal leucine recognition by class I PDZ domains

The Na(+)/H(+) exchanger regulatory factor (NHERF; also known as EBP50) contains two PDZ domains that mediate the assembly of transmembrane and cytosolic proteins into functional signal transduction complexes. The NHERF PDZ1 domain interacts specifically with the motifs DSLL, DSFL, and DTRL present at the carboxyl termini of the beta(2) adrenergic receptor (beta(2)AR), the platelet-derived growth factor receptor (PDGFR), and the cystic fibrosis transmembrane conductance regulator (CFTR), respectively, and plays a central role in the physiological regulation of these proteins. The crystal structure of the human NHERF PDZ1 has been determined at 1.5 A resolution using multiwavelength anomalous diffraction phasing. The overall structure is similar to known PDZ structures, with notable differences in the NHERF PDZ1 carboxylate-binding loop that contains the GYGF motif, and the variable loop between the beta2 and beta3 strands. In the crystalline state, the carboxyl-terminal sequence DEQL of PDZ1 occupies the peptide-binding pocket of a neighboring PDZ1 molecule related by 2-fold crystallographic symmetry. This structure reveals the molecular mechanism of carboxyl-terminal leucine recognition by class I PDZ domains, and provides insights into the specificity of NHERF interaction with the carboxyl termini of several membrane receptors and ion channels, including the beta(2)AR, PDGFR, and CFTR.     

The venerable inveterate invertebrate TRP channels

The transient receptor potential (TRP) superfamily is subdivided into four main classes of cation channels, TRPC, TRPV, TRPM and TRPN, each of which includes members in worms, flies, mice and humans. While the biophysical features of many of the mammalian channels have been described, relatively little is known concerning the biological roles of these channels. Forward genetic screens in Drosophila melanogaster and Caenorhabditis elegans have led to the identification of the founding members of each of these four subfamilies. Moreover, phenotypic analyses of invertebrate mutants have contributed greatly to our understanding of the roles of TRP proteins. A recurring theme is that many of these proteins function in sensory signaling processes ranging from vision to olfaction, osmosensation, light touch, social feeding, and temperature- and mechanically-induced nociception. In addition, at least one invertebrate TRP protein is required for cell division. As many of these functions may be conserved among the mammalian TRPs, the invertebrate TRPs offer valuable genetic handles for characterizing the functions of these cation channels in vivo.     

Regulation of phospholipase C-beta 3 activity by Na+/H+ exchanger regulatory factor 2

Among the phospholipase C that catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate, four mammalian phospholipase C-beta (PLC-beta) isotypes (isotypes 1-4) are activated through G protein-coupled receptors (GPCRs). Although the regulation of the PLC-betas by GPCRs and heterotrimeric G proteins has been extensively studied, little is known about the molecular determinants that regulate their activity. The PLC-beta isozymes carry a putative PSD-95/Dlg/ZO-1 (PDZ) binding motif (X(S/T)X(V/L)COOH) at their carboxyl terminus, which is implicated in specific interactions with anchor proteins. Using the yeast two-hybrid system, we identified Na(+)/H(+) exchanger regulatory factor 2 (NHERF2) as a protein that interacted with a C-terminal heptapeptide of PLC-beta3. Immunoprecipitation studies revealed that NHERF2 interacts specifically with PLC-beta3, but not with other PLC-beta isotypes. Furthermore, PLC-beta3 interacted with NHERF2 rather than with other PDZ-containing proteins. This interaction required the COOH-terminal NTQL sequence of PLC-beta3 and the second PDZ domain of NHERF2. Interestingly, NHERF2 potentiated the PLC-beta activation by carbachol in COS7 and HeLa cells, while mutant NHERF2, lacking the second PDZ domain, had no such effect. Taken together, the data suggest that NHERF2 may act as a modulator underlying the process of PLC-beta3-mediated signaling.