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.     

Concerted actions of NHERF2 and WNK4 in regulating TRPV5

With-no-lysine (K) kinase 4 (WNK4) is a protein serine/threonine kinase associated with a Mendelian form of hypertension. WNK4 is an integrative regulator of renal transport of Na⁺, K⁺, and Cl⁻ as shown in Xenopus oocyte system. In addition, WNK4 enhances the surface expression of epithelial Ca²⁺ channel TRPV5, which plays a key role in the fine tuning of renal Ca²⁺ reabsorption. Variations in the magnitude of WNK4-mediated regulation on TRPV5 in Xenopus oocytes suggest additional cellular components with limited expression are required for the regulation. In this study, we identified the Na⁺/H⁺ exchanger regulating factor 2 (NHERF2) as a critical component for the positive regulation of TRPV5 by WNK4. NHERF2 augmented the positive effect of WNK4 on TRPV5, whereas its homolog NHERF1 had no effect when tested in the Xenopus oocyte system. The C-terminal PDZ binding motif of TRPV5 was required for the regulation by NHERF2. While NHERF2 interacted with TRPV5, no association between NHERF2 and WNK4 was detected using a GST pull-down assay. WNK4 increased the forward trafficking of TRPV5; however, it also caused an accelerated decline of the functional TRPV5 channels at later stage of co-expression. NHERF2 stabilized TRPV5 at the plasma membrane without interrupting the forward trafficking of TRPV5, thus prevented the decline of functional TRPV5 channel caused by WNK4 at later stage. The complementary and orderly regulations of WNK4 and NHERF2 allow TRPV5 functions at higher level for a longer period to maximize Ca²⁺ influx.     

TRPC4 forms store-operated Ca2+ channels in mouse mesangial cells

Studies were performed to identify the molecular component responsible for store-operated Ca²⁺ entry in murine mesangial cells (MMC). Because the canonical transient receptor potential (TRPC) family of proteins was previously shown to comprise Ca²⁺-selective and -nonselective cation channels in a variety of cells, we screened TRPC1–TRPC7 with the use of molecular methods and the fura 2 method to determine their participation as components of the mesangial store-operated Ca²⁺ (SOC) channel. Using TRPC-specific primers and RT-PCR, we found that cultured MMC contained mRNA for TRPC1 and TRPC4 but not for TRPC2, TRPC3, TRPC5, TRPC6, and TRPC7. Immunocytochemical staining of MMC revealed predominantly cytoplasmic expression of TRPC1 and plasmalemmal expression of TRPC4. The role of TRPC4 in SOC was determined with TRPC4 antisense and fura 2 ratiometric measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i). SOC was measured as the increase in [Ca²⁺]_i after extracellular Ca²⁺ was increased from <10 nM to 1 mM in the continued presence of thapsigargin. We found that TRPC4 antisense, which reduced plasmalemmal expression of TRPC4, inhibited SOC by 83%. Incubation with scrambled TRPC4 oligonucleotides did not affect SOC. Immunohistochemical staining identified expressed TRPC4 in the glomeruli of mouse renal sections. The results of RT-PCR performed to distinguish between TRPC4- and TRPC4- were consistent with expression of both isoforms in brain but with only TRPC4- expression in MMC. These studies show that TRPC4- may form the homotetrameric SOC in mouse mesangial cells. canonical transient receptor potential; TRPC4-; TRPC4-; TRPC1; fura 2; glomerulus     

Plasticity of TRPC expression in arterial smooth muscle: correlation with store-operated Ca2+ entry

Loss of the smooth muscle contractile phenotype is critical in atherosclerosis and in restenosis after angioplasty, but its early signals are incompletely understood. In this study, we have explored the role of transient receptor potential canonical (TRPC) proteins, which have been suggested to mediate store-operated Ca²⁺ entry (SOCE). Contractility of rat cerebral arteries in organ culture is preserved for several days, whereas SOCE is increased. In correlation with this increase is that nifedipine-insensitive whole cell current, activated by depletion of intracellular Ca²⁺ stores, was increased by 50% in cells isolated from arteries cultured for 3 days. TRPC1 and TRPC6 mRNA were more than fivefold increased in cells isolated after organ culture, whereas TRPC3 was decreased. Immunofluorescent staining and/or Western blotting of arteries and isolated cells showed upregulation of TRPC1 and TRPC6 proteins during organ culture. In intact arteries, TRPC4 expression correlated with the amount of endothelium present. Ca²⁺ addition after store depletion caused a contraction in cultured, but not in freshly dissected, arteries. A polyclonal TRPC1 antibody directed against an extracellular epitope inhibited this contraction by 50%. To investigate the basis of the TRPC upregulation and assess its possible clinical significance, segments of human internal mammary artery were organ cultured for 24 h and then exposed to balloon dilatation in vitro, followed by further culturing for up to 48 h. After dilatation, TRPC1 and TRPC6 mRNA were progressively increased compared with undilated control segments. The results of this study indicate that vascular injury enhances plasticity in TRPC expression, that TRPC expression correlates with cellular Ca²⁺ handling, and that TRPC1 is a subunit of upregulated store-operated Ca²⁺ channels. differentiation; ion channels; angioplasty; organ culture     

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.     

The Protein Scaffold NHERF-1 Controls the Amplitude and Duration of Localized Protein Kinase D Activity

Protein kinase D (PKD) transduces an abundance of signals downstream of diacylglycerol production. The mammalian PKD family consists of three isoforms, PKD1, PKD2, and PKD3; of these PKD1 and PKD2 contain PDZ-binding motifs at their carboxyl termini. Here we show that membrane-localized NHERF scaffold proteins provide a nexus for tightly controlled PKD signaling via a PDZ domain interaction. Using a proteomic array containing 96 purified PDZ domains, we have identified the first PDZ domain of NHERF-1 as an interaction partner for the PDZ-binding motifs of both PKD1 and PKD2. A fluorescence resonance energy transfer-based translocation assay reveals a transient association of PKD1 and PKD2 with NHERF-1 in live cells that is triggered by phorbol ester stimulation and, importantly, differs strikingly from the sustained translocation to plasma membrane. Targeting a fluorescence resonance energy transfer-based kinase activity reporter for PKD to NHERF scaffolds reveals a unique signature of PKD activation at the scaffold that is distinct from that of general cytosolic or plasma membrane activity. Specifically, agonist-evoked activation of PKD at the scaffold is rapid and sustained but blunted in magnitude when compared with cytosolic PKD. Thus, live cell imaging of PKD activity demonstrates ultrasensitive control of kinase signaling at the scaffold compared with bulk activity in the cytosol or at the plasma membrane.Protein kinase D (PKD)² plays a role in numerous processes including cell proliferation, cell survival, immune cell signaling, gene expression, vesicle trafficking, and neuronal development (1). The PKD family consists of three members belonging to the Ca²⁺/calmodulin-dependent kinase group of serine/threonine protein kinases. Each isoform contains a conserved catalytic core and an amino-terminal regulatory moiety. This regulatory region contains two cysteine-rich (C1) domains and a pleckstrin homology domain that autoinhibits the kinase (2). The C1 domains are membrane-targeting modules that bind diacylglycerol (DAG) and its functional analogues, phorbol esters, thus recruiting PKD to membranes (3). The PKD1 and PKD2 isoforms additionally contain PDZ-binding motifs at their carboxyl termini that can target the kinases to distinct subcellular scaffolds through interactions with PDZ domain-containing proteins (4).PKD transduces signals downstream of the second messenger DAG. In addition to membrane recruitment by DAG, activation of PKD requires phosphorylation by novel protein kinase C (PKC) family members at two sites within its catalytic core (5, 6). The novel PKCs themselves contain C1 domains and are allosterically activated by DAG-mediated membrane binding; thus, DAG production leads to PKD activation through coincident activation of the novel PKCs and localization of PKD near its upstream kinases. Hence, activation of phospholipase C (PLC)-coupled receptors (such as certain G protein-coupled receptors (GPCRs) or receptor tyrosine kinases) results in the production of second messengers including DAG, and this leads to recruitment and activation of the novel PKCs and thus also PKD.PDZ (PSD-95, Discs large, ZO-1) domains are compact, globular structures of ∼90 residues, occurring in one or multiple copies within a protein, that mediate protein-protein interactions (7). These interactions occur via binding to other PDZ domains or, more commonly, by recognition of short amino acid motifs in the carboxyl termini of target proteins commonly terminating in a hydrophobic residue (8). In the case of PKD1 and PKD2, the last four amino acids are VSIL and ISVL, respectively. Here we identify Na⁺/H⁺ exchanger regulatory factor 1 (NHERF-1) as a PDZ domain-containing protein that interacts with the PDZ-binding motif of both PKD1 and PKD2.NHERF-1 was originally cloned as a critical protein component for the inhibition of Na⁺/H⁺ exchanger 3 by protein kinase A (9). NHERF-1 is 52% identical to NHERF-2, a family member with which it shares the conserved domain structure of two PDZ domains followed by an ezrin-radixin-moesin (ERM)-binding region (10). Parallel studies demonstrating its ability to strongly interact with ezrin independently identified NHERF-1 as ERM-binding phosphoprotein 50 (11). Via this ERM-binding region, NHERF-1 and NHERF-2 are predominantly localized near the actin cytoskeleton, thus poising them near the plasma membrane where they function as scaffolds. Since these original cloning reports, numerous studies have identified over 30 binding partners of these scaffold proteins including GPCRs, tyrosine kinase receptors, other adaptor proteins, signaling enzymes, and ion channels (12, 13).Here we identify PKD1 and PKD2 as NHERF-1-interacting proteins. Using a fluorescence resonance energy transfer (FRET)-based assay to assess molecular proximity, both PKD1 and PKD2 are shown to transiently associate with NHERF-1 following PKD activation. Furthermore, through use of genetically encoded reporters for PKD activity, we show a unique signature of PKD activation at the NHERF scaffold. Specifically, signaling is more tightly regulated at the scaffold than in the cytosol or bulk plasma membrane. Phosphatase activity is higher at NHERF than at the plasma membrane, resulting in a more rapidly reversible PKD response at the scaffold, and following an agonist-evoked response, PKD signaling is prolonged compared with the length of response in the cytosol. Our data identify NHERF-1 as a novel nexus of PKD signaling and raise the possibility that PKD may act as a novel regulator of proteins at the NHERF scaffold.     

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

To investigate thepossible role of members of the mammalian transient receptor potential(TRP) channel family (TRPC1-7) in vasoconstrictor-inducedCa²⁺ entry in vascular smooth muscle cells, we studied[Arg⁸]-vasopressin (AVP)-activated channels in A7r5aortic smooth muscle cells. AVP induced an increase in free cytosolicCa²⁺ concentration ([Ca²⁺]_i)consisting of Ca²⁺ release and Ca²⁺ influx.Whole cell recordings revealed the activation of a nonselective cationcurrent with a doubly rectifying current-voltage relation strikinglysimilar to those described for some heterologously expressed TRPCisoforms. The current was also stimulated by direct activation of Gproteins as well as by activation of the phospholipase C-coupledplatelet-derived growth factor receptor. Currents were not activated bystore depletion or increased [Ca²⁺]_i.Application of 1-oleoyl-2-acetyl-sn-glycerol stimulated the current independently of protein kinase C, a characteristic property ofthe TRPC3/6/7 subfamily. Like TRPC6-mediated currents, cation currentsin A7r5 cells were increased by flufenamate. Northern hybridizationrevealed mRNA coding for TRPC1 and TRPC6. We therefore suggest thatTRPC6 is a molecular component of receptor-stimulated Ca²⁺-permeable cation channels in A7r5 smooth muscle cells.

     

Oligomerization of NHERF-1 and NHERF-2 PDZ domains: differential regulation by association with receptor carboxyl-termini and by phosphorylation.

PDZ domains bind to the carboxyl-termini of target proteins, and some PDZ domains are capable of oligomerization to facilitate the formation of intracellular signaling complexes. The Na(+)/H(+) exchanger regulatory factor (NHERF-1; also called "EBP50") and its relative NHERF-2 (also called "E3KARP", "SIP-1", and "TKA-1") both have two PDZ domains. We report here that the PDZ domains of NHERF-1 and NHERF-2 bind specifically to each other but not to other PDZ domains. Purified NHERF-2 PDZ domains associate with each other robustly in the absence of any associated proteins, but purified NHERF-1 PDZ domains associate with each other only weakly when examined alone. The oligomerization of the NHERF-1 PDZ domains is greatly facilitated when they are bound with carboxyl-terminal ligands, such as the carboxyl-termini of the beta(2)-adrenergic receptor or the platelet-derived growth factor receptor. Oligomerization of full-length NHERF-1 is also enhanced by mutation of serine 289 to aspartate (S289D), which mimics the phosphorylated form of NHERF-1. Co-immunoprecipitation experiments with differentially tagged versions of the NHERF proteins reveal that NHERF-1 and NHERF-2 form homo- and hetero-oligomers in a cellular context. A point-mutated version of NHERF-1 (S289A), which cannot be phosphorylated on serine 289, exhibits a reduced capacity for co-immunoprecipitation from cells. These studies reveal that both NHERF-1 and NHERF-2 can oligomerize, which may facilitate NHERF-mediated formation of cellular signaling complexes. These studies furthermore reveal that oligomerization of NHERF-1, but not NHERF-2, is highly regulated by association with other proteins and by phosphorylation.     

NHERF2/NHERF3 Protein Heterodimerization and Macrocomplex Formation Are Required for the Inhibition of NHE3 Activity by Carbachol

NHERF1, NHERF2, and NHERF3 belong to the NHERF (Na⁺/H⁺ exchanger regulatory factor) family of PSD-95/Discs-large/ZO-1 (PDZ) scaffolding proteins. Individually, each NHERF protein has been shown to be involved in the regulation of multiple receptors or transporters including Na⁺/H⁺ exchanger 3 (NHE3). Although NHERF dimerizations have been reported, results have been inconsistent, and the physiological function of NHERF dimerizations is still unknown. The current study semiquantitatively compared the interaction strength among all possible homodimerizations and heterodimerizations of these three NHERF proteins by pulldown and co-immunoprecipitation assays. Both methods showed that NHERF2 and NHERF3 heterodimerize as the strongest interaction among all NHERF dimerizations. In vivo NHERF2/NHERF3 heterodimerization was confirmed by FRET and FRAP (fluorescence recovery after photobleach). NHERF2/NHERF3 heterodimerization is mediated by PDZ domains of NHERF2 and the C-terminal PDZ domain recognition motif of NHERF3. The NHERF3-4A mutant is defective in heterodimerization with NHERF2 and does not support the inhibition of NHE3 by carbachol. This suggests a role for NHERF2/NHERF3 heterodimerization in the regulation of NHE3 activity. In addition, both PDZ domains of NHERF2 could be simultaneously occupied by NHERF3 and another ligand such as NHE3, α-actinin-4, and PKCα, promoting formation of NHE3 macrocomplexes. This study suggests that NHERF2/NHERF3 heterodimerization mediates the formation of NHE3 macrocomplexes, which are required for the inhibition of NHE3 activity by carbachol.     

Canonical transient receptor potential TRPC7 can function as both a receptor- and store-operated channel in HEK-293 cells

Previous studies on the activation mechanism of canonical transient receptor potential (TRPC) channels have often produced conflicting conclusions. All seven have been shown to be activated by phospholipase C (PLC)-coupled receptors, but TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, and TRPC7 have also been proposed to function as store-operated channels.¹ In the case of TRPC3, the expression environment and the expression level appear to determine the mode of regulation. Evidence of a close structural relative of TRPC3, TRPC7, has been presented that this channel is activated by receptor activation or by store depletion. On the basis of previous findings for TRPC3, we reasoned that subtle differences in structure or expression conditions might account for the apparent distinct gating mechanisms of TRPC7. To reexamine the mode of activation of TRPC7, we stably and transiently transfected human embryonic kidney (HEK)-293 cells with cDNA encoding for human TRPC7. We examined the ability of a PLC-activating agonist and an intracellular Ca²⁺ store-depleting agent to activate these channels. Our findings demonstrate that when transiently expressed in HEK-293 cells, TRPC7 forms channels that are activated by PLC-stimulating agonists, but not by Ca²⁺ store depletion. However, when stably expressed in HEK-293 cells, TRPC7 can be activated by either Ca²⁺ store depletion or PLC activation. To our knowledge, this is the first demonstration of a channel protein that can be activated by both receptor- and store-operated modes in the same cell. In addition, the results reconcile the apparently conflicting findings of other laboratories regarding TRPC7 regulation. calcium signaling; nonselective cation channels     

Binding to Na+/H+ exchanger regulatory factor 2 (NHERF2) affects trafficking and function of the enteropathogenic Escherichia coli type III secretion system effectors Map,EspI and NleH

Enteropathogenic Escherichia coli (EPEC) strains are diarrhoeal pathogens that use a type III secretion system to translocate effector proteins into host cells in order to colonize and multiply in the human gut. Map, EspI and NleH1 are conserved EPEC effectors that possess a C‐terminal class I PSD‐95/Disc Large/ZO‐1 (PDZ)‐binding motif. Using a PDZ array screen we identified Na⁺/H⁺ exchanger regulatory factor 2 (NHERF2), a scaffold protein involved in tethering and recycling ion channels in polarized epithelia that contains two PDZ domains, as a common target of Map, EspI and NleH1. Using recombinant proteins and co‐immunoprecipitation we confirmed that NHERF2 binds each of the effectors. We generated a HeLa cell line stably expressing HA‐tagged NHERF2 and found that Map, EspI and NleH1 colocalize and interact with intracellular NHERF2 via their C‐terminal PDZ‐binding motif. Overexpression of NHERF2 enhanced the formation and persistence of Map‐induced filopodia, accelerated the trafficking of EspI to the Golgi and diminished the anti‐apoptotic activity of NleH1. The binding of multiple T3SS effectors to a single scaffold protein is unique. Our data suggest that NHERF2 may act as a plasma membrane sorting site, providing a novel regulatory mechanism to control the intracellular spatial and temporal effector protein activity.     

Role of the scaffold protein RACK1 in apical expression of CFTR

Previous studies from this laboratory demonstrated a role for protein kinase C (PKC) in the regulation of cAMP-dependent cystic fibrosis transmembrane regulator (CFTR) Cl channel function via binding of PKC to RACK1, a receptor for activated C kinase, and of RACK1 to human Na⁺/H⁺ exchanger regulatory factor (NHERF1). In the present study, we investigated the role of RACK1 in regulating CFTR function in a Calu-3 airway epithelial cell line. Confocal microscopy and biotinylation of apical surface proteins demonstrate apical localization of RACK1 independent of actin. Mass spectrometric analysis of NHERF1 revealed copurification of tubulin, which, in in vitro binding assays, selectively binds to NHERF1, but not RACK1, via a PDZ1 domain. In binding and pulldown assays, we show direct binding of a PDZ2 domain to NHERF1, pulldown of endogenous NHERF1 by a PDZ2 domain, and inhibition of NHERF1-tubulin binding by a PDZ1 domain. Downregulation of RACK1 using double-stranded silencing RNA reduced the amount of RACK1 by 77.5% and apical expression of biotinylated CFTR by 87.4%. Expression of CFTR, NHERF1, and actin were not altered by treatment with siRACK1 or by nontargeting control silencing RNA, which, in addition, did not affect RACK1 expression. On the basis of these results, we model a RACK1 proteome consisting of PKC-RACK1-NHERF1-NHERF1-tubulin with a role in stable expression of CFTR in the apical plasma membrane of epithelial cells. silencing RNA; downregulation; biotinylation; tubulin; NHERF1; tailless cystic fibrosis transmembrane regulator; PDZ domain     

Opposite regulatory effects of TRPC1 and TRPC5 on neurite outgrowth in PC12 cells

The transient receptor potential (TRPC) family of Ca^2 + permeable, non-selective cation channels is abundantly expressed in the brain, and can function as store-operated (SOC) and store-independent channels depending on their interaction with the ER Ca^2 + sensor STIM1. TRPC1 and TRPC5 have critical roles in neurite outgrowth, however which of their functions regulate neurite outgrowth is unknown. In this study, we investigated the effects of TRPC channels and their STIM1-induced SOC activity on neurite outgrowth of PC12 cells. We report that PC12 cell differentiation down-regulates TRPC5 expression, whereas TRPC1 expression is retained. TRPC1 and TRPC5 interact with STIM1 through the STIM1 ERM domain. Transfection of TRPC1 and TRPC5 increased the receptor-activated Ca^2 + influx that was markedly augmented by the co-expression of STIM1. Topical expression of TRPC1 in PC12 cells markedly increased neurite outgrowth while that of TRPC5 suppressed neurite outgrowth. Suppression of neurite outgrowth by TRPC5 requires the channel function of TRPC5. However, strikingly, multiple lines of evidence show that the TRPC1-induced neurite outgrowth was independent of TRPC1-mediated Ca^2 + influx. Thus, a) TRPC1 and TRPC5 similarly increased Ca^2 + influx but only TRPC1 induced neurite outgrowth, b) the constitutively STIM1^D76A mutant that activates Ca^2 + influx by TRPC and Orai channels did not increase neurite outgrowth, c) co-expression of TRPC5 with TRPC1 suppressed the effect of TRPC1 on neurite outgrowth, d) and most notable, channel-dead pore mutant of TRPC1 increased neurite outgrowth to the same extent as TRPC1^WT. Suppression of TRPC1-induced neurite outgrowth by TRPC5 was due to a marked reduction in the surface expression of TRPC1. We conclude that the regulation of neurite outgrowth by TRPC1 is independent of Ca^2 + influx and TRPC1-promoted neurite outgrowth depends on the surface expression of TRPC1. It is likely that TRPC1 acts as a scaffold at the cell surface to assemble a signaling complex to stimulate neurite outgrowth.     

Functional significance of channels and transporters expressed in the inner ear and kidney

A number of ion channels and transporters are expressed in both the inner ear and kidney. In the inner ear, K⁺ cycling and endolymphatic K⁺, Na⁺, Ca²⁺, and pH homeostasis are critical for normal organ function. Ion channels and transporters involved in K⁺ cycling include K⁺ channels, Na⁺-2Cl^–-K⁺ cotransporter, Na⁺/K⁺-ATPase, Cl^– channels, connexins, and K⁺/Cl^– cotransporters. Furthermore, endolymphatic Na⁺ and Ca²⁺ homeostasis depends on Ca²⁺-ATPase, Ca²⁺ channels, Na⁺ channels, and a purinergic receptor channel. Endolymphatic pH homeostasis involves H⁺-ATPase and Cl^–/HCO₃^– exchangers including pendrin. Defective connexins (GJB2 and GJB6), pendrin (SLC26A4), K⁺ channels (KCNJ10, KCNQ1, KCNE1, and KCNMA1), Na⁺-2Cl^–-K⁺ cotransporter (SLC12A2), K⁺/Cl^– cotransporters (KCC3 and KCC4), Cl^– channels (BSND and CLCNKA + CLCNKB), and H⁺-ATPase (ATP6V1B1 and ATPV0A4) cause hearing loss. All these channels and transporters are also expressed in the kidney and support renal tubular transport or signaling. The hearing loss may thus be paralleled by various renal phenotypes including a subtle decrease of proximal Na⁺-coupled transport (KCNE1/KCNQ1), impaired K⁺ secretion (KCNMA1), limited HCO₃^– elimination (SLC26A4), NaCl wasting (BSND and CLCNKB), renal tubular acidosis (ATP6V1B1, ATPV0A4, and KCC4), or impaired urinary concentration (CLCNKA). Thus, defects of channels and transporters expressed in the kidney and inner ear result in simultaneous dysfunctions of these seemingly unrelated organs. cochlea; vestibular labyrinth; stria vascularis; deafness; renal tubule     

Role of a PDZ1 domain of NHERF1 in the binding of airway epithelial RACK1 to NHERF1

In past studies, we demonstrated regulation of CFTR Cl channel function by protein kinase C (PKC)- through the binding of PKC- to RACK1 (a receptor for activated C-kinase) and of RACK1 to human Na⁺/H⁺ exchanger regulatory factor (NHERF1). In this study, we investigated the site of RACK1 binding on NHERF1 using solid-phase and solution binding assays and pulldown, immunoprecipitation, and ³⁶Cl efflux experiments. Recombinant RACK1 binding to glutathione S-transferase (GST)-tagged PDZ1 domain of NHERF1 was 10-fold higher than its binding to GST-tagged PDZ2 domain of NHERF1. PDZ1 binds to RACK1 in a dose-dependent manner and vice versa, with similar binding constants of 1.67 and 1.26 µg, respectively. Interaction of the PDZ1 domain with RACK1 was not blocked by binding of activated PKC- to RACK1. A GST-tagged PDZ1 domain pulled down endogenous RACK1 from Calu-3 cell lysate. An internal 11-amino acid motif embedding the GYGF carboxylate binding loop of PDZ1 binds to RACK1, inhibits binding of recombinant NHERF1 and RACK1, pulls down endogenous RACK1 from Calu-3 cell lysate, and blocks coimmunoprecipitation of endogenous RACK1 with endogenous NHERF1 but does not affect cAMP-dependent activation of CFTR. A similar amino acid sequence in the PDZ2 domain did not bind RACK1. Our results indicate binding of Calu-3 RACK1 predominantly to the PDZ1 domain of NHERF1 at a site encompassing the GYGF loop of the PDZ1 domain and a site on RACK1 distinct from a PKC- binding site. CFTR activation by cAMP-generating agent is not affected by loss of RACK1-NHERF1 interaction. cystic fibrosis; cystic fibrosis transmembrane conductance regulator; protein-protein interaction; slot blot assay; pulldown; PDZ domain; chloride efflux; immunoprecipitation     

TRP expression pattern and the functional importance of TRPC3 in primary human T-cells

TRP proteins form ion channels which are activated following receptor stimulation. In T-cell lines, expression data of TRP proteins have been published. However, almost no data about TRP expression is available in primary human T-cells. Using RT-PCR and quantitative RT-PCR, we compare the expression of TRP mRNA in 1) human peripheral blood lymphocytes, which are a mix of mostly mono-nuclear blood lymphocytes but contain other leucocytes, 2) a pure human CD4⁺ T-helper cell population in the resting (= naïve) and activated (= effector) state, and 3) two commonly used CD4⁺ Jurkat T-cell lines, E6-1 and parental. To mimic physiological cell stimulation, we analyzed TRP expression in primary human cells in a quantitative way over several days following formation of an immunological synapse through stimulation with antibody-coated beads. The TRP expression profile of primary human T-cells was significantly different from Jurkat T-cells. Among the TRP mRNAs of the TRPC, TRPM, and TRPV family, we found consistent expression of TRPC1, TRPC3, TRPV1, TRPM2, and TRPM7 in primary human CD4⁺ T-cells of all analyzed blood donors. Among these, TRPC3 and TRPM2 were strongly up-regulated following stimulation, but with different kinetics. We found that TRPC3 modulates Ca²⁺-dependent proliferation of primary CD4⁺ T-cells indicating that TRPC3 may be involved in Ca²⁺ homeostasis in T-cells besides the well-established STIM and ORAI proteins which are responsible for store-operated Ca²⁺ entry.     

Ezrin Induces Long-Range Interdomain Allostery in the Scaffolding Protein NHERF1

Scaffolding proteins are molecular switches that control diverse signaling events. The scaffolding protein Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) assembles macromolecular signaling complexes and regulates the macromolecular assembly, localization, and intracellular trafficking of a number of membrane ion transport proteins, receptors, and adhesion/antiadhesion proteins. NHERF1 begins with two modular protein-protein interaction domains—PDZ1 and PDZ2—and ends with a C-terminal (CT) domain. This CT domain binds to ezrin, which, in turn, interacts with cytosekeletal actin. Remarkably, ezrin binding to NHERF1 increases the binding capabilities of both PDZ domains. Here, we use deuterium labeling and contrast variation neutron-scattering experiments to determine the conformational changes in NHERF1 when it forms a complex with ezrin. Upon binding to ezrin, NHERF1 undergoes significant conformational changes in the region linking PDZ2 and its CT ezrin-binding domain, as well as in the region linking PDZ1 and PDZ2, involving very long range interactions over 120 Å. The results provide a structural explanation, at mesoscopic scales, of the allosteric control of NHERF1 by ezrin as it assembles protein complexes. Because of the essential roles of NHERF1 and ezrin in intracellular trafficking in epithelial cells, we hypothesize that this long-range allosteric regulation of NHERF1 by ezrin enables the membrane-cytoskeleton to assemble protein complexes that control cross-talk and regulate the strength and duration of signaling.     

Defective coupling of apical PTH receptors to phospholipase C prevents internalization of the Na+-phosphate cotransporter NaPi-IIa in Nherf1-deficient mice

Phosphate reabsorption in the renal proximal tubule occurs mostly via the type IIa Na⁺-phosphate cotransporter (NaP_i-IIa) in the brush border membrane (BBM). The activity and localization of NaP_i-IIa are regulated, among other factors, by parathyroid hormone (PTH). NaP_i-IIa interacts in vitro via its last three COOH-terminal amino acids with the PDZ protein Na⁺/H⁺-exchanger isoform 3 regulatory factor (NHERF)-1 (NHERF1). Renal phosphate reabsorption in Nherf1-deficient mice is altered, and NaP_i-IIa expression in the BBM is reduced. In addition, it has been proposed that NHERF1 and NHERF2 are important for the coupling of PTH receptors (PTHRs) to phospholipase C (PLC) and the activation of the protein kinase C pathway. We tested the role of NHERF1 in the regulation of NaP_i-IIa by PTH in Nherf1-deficient mice. Immunohistochemistry and Western blotting demonstrated that stimulation of apical and basolateral receptors with PTH-(1–34) led to internalization of NaP_i-IIa in wild-type and Nherf1-deficient mice. Stimulation of only apical receptors with PTH-(3–34) failed to induce internalization in Nherf1-deficient mice. Expression and localization of apical PTHRs were similar in wild-type and Nherf1-deficient mice. Activation of the protein kinase C- and A-dependent pathways with 1,2-dioctanoyl-sn-glycerol or 8-bromo-cAMP induced normal internalization of NaP_i-IIa in wild-type, as well as Nherf1-deficient, mice. Stimulation of PLC activity due to apical PTHRs was impaired in Nherf1-deficient mice. These data suggest that NHERF1 in the proximal tubule is important for PTH-induced internalization of NaP_i-IIa and, specifically, couples the apical PTHR to PLC. phosphate cotransporter; PDZ protein; parathyroid hormone; proximal tubule     

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.